Estrogen receptor β and progesterone receptor mRNA in the intergeniculate leaflet of the female rat

The present study was undertaken to explore the possibility that the integration of hormonal cues in the regulation of neuroendocrine mechanisms may occur outside of the hypothalamus at the level of the lateral geniculate body. In situ hybridization for mRNA encoding estrogen receptor β and progesterone receptor was carried out on sections containing the lateral geniculate body using [³⁵S]-labeled antisense riboprobes. Labeled cells were present in different limbic and hypothalamic sites as described previously. Populations of cells distributed homogeneously in the ventral lateral geniculate nucleus and intergeniculate leaflet were also found to express mRNA for estrogen receptor β and progesterone receptor. The dorsal lateral geniculate nucleus lacked specific labeling for either type of gonadal steroid hormone receptor mRNA. The present observation together with the recent demonstration of a direct pathway between the intergeniculate leaflet and hypothalamic neuroendocrine cells indicate that integration of hormonal and photic stimuli in the central regulation of endocrine mechanisms occurs outside of the hypothalamus in the lateral geniculate body.     

Dorsal raphe nucleus modulates neuronal activity in rat intergeniculate leaflet

Serotonergic input from midbrain raphe nuclei is believed to have a significant effect on mammalian circadian timing system. The suprachiasmatic nucleus (SCN) receives its serotonergic input from the median raphe nucleus, while the intergeniculate leaflet (IGL) receives serotonergic innervation from the dorsal raphe nucleus (DRN). The present paper was aimed at determining whether projection from the DRN affected rhythmic neuronal oscillations in the IGL of rats. We investigated the impact of electrolytic lesions and electric stimulation of the DRN on spontaneous isoperiodic (i.e. burst firing with a constant interburst interval) neuronal activity recorded in the IGL. In all our experiments a complete lesion of the DRN always caused a significant increase (ca. 100%) of spontaneous activity of IGL neurons, their oscillatory character having been maintained, though. On the other hand, electric stimulation of the DRN produced a transient decrease in firing rate oscillations of the IGL neurons. The obtained results indicate that the neuronal projection from the DRN has a substantial modulating effect on IGL activity-an important element of the mechanism of the circadian time-keeping system that mediates the transfer of non-photic information to the SCN by modulating its activity. The observed increase of isoperiodic activity in the IGL after DRN lesion and a transient decrease in this activity after electric stimulation indicate an inhibitory character of this effect. The present findings corroborate the hypothesis that the DRN is a one of the major and extremely important source of the modulatory inputs to the mammalian circadian time-keeping system.     

Distribution of delta opioid receptor immunoreactivity in the hamster suprachiasmatic nucleus and intergeniculate leaflet

The hamster suprachiasmatic nucleus (SCN) is innervated by a dense plexus of enkephalin-containing axons originating from cells in the intergeniculate leaflet (IGL) of the thalamus. However, the distribution of opioid receptors within the hamster SCN has not been reported. Opioid receptors consist of three primary subtypes: mu, delta and kappa opioid receptors. Enkephalins have the highest affinity for delta opioid receptors. Therefore, in the present study, we examined the distribution of delta opioid receptor immunoreactivity in the hamster SCN and the IGL of the thalamus. Coronal sections of the hamster hypothalamus inclusive of the SCN or thalamic regions containing the IGL were prepared at specific times of the day and labeled with anti-delta opioid receptor polyclonal antisera using standard immunohistochemical techniques. delta opioid receptors were heavily distributed within rostral-caudal regions of the SCN, with the densest labeling located in the ventral and medial regions of the mid-SCN. Similar patterns of labeling were observed for tissue prepared during mid-day or mid-night times. In contrast, delta opioid receptor immunoreactivity only sparsely labeled cells in the IGL. Cellular staining in all regions appeared as dark punctate labeling surrounding cells, indicative of terminal boutons. Therefore, it is suggested that delta opioid receptors are located presynaptically on axon terminals within the hamster SCN and IGL. These results suggest that delta opioid receptors may play a role in modulating circadian rhythms generated within the SCN, possibly by regulating transmitter release within the nucleus.     

Efferent projections of the intergeniculate leaflet and the ventral lateral geniculate nucleus in the rat

The intergeniculate leaflet (IGL) and the ventral lateral geniculate nucleus (VLG) are ventral thalamic derivatives within the lateral geniculate complex. In this study, IGL and VLG efferent projections were compared by using anterograde transport of Phaseolus vulgaris-leucoagglutinin and retrograde transport of FluoroGold. Projections from the IGL and VLG leave the geniculate in four pathways. A dorsal pathway innervates the thalamic lateral dorsal nucleus (VLG), the reuniens and rhomboid nuclei (VLG and IGL), and the paraventricular nucleus (IGL). A ventral pathway runs through the geniculohypothalamic tract to the suprachiasmatic nucleus and the anterior hypothalamus (IGL). A medial pathway innervates the zona incerta and dorsal hypothalamus (VLG and IGL); the lateral hypothalamus and perifornical area (VLG); and the retrochiasmatic area (RCA), dorsomedial hypothalamic nucleus, and subparaventricular zone (IGL). A caudal pathway projects medially to the posterior hypothalamic area and periaqueductal gray and caudally along the brachium of the superior colliculus to the medial pretectal area and the nucleus of the optic tract (IGL and VLG). Caudal IGL axons also terminate in the olivary pretectal nucleus, the superficial gray of the superior colliculus, and the lateral and dorsal terminal nuclei of the accessory optic system. Caudal VLG projections innervate the lateral posterior nucleus, the anterior pretectal nucleus, the intermediate and deep gray of the superior colliculus, the dorsal terminal nucleus, the midbrain lateral tegmental field, the interpeduncular nucleus, the ventral pontine reticular formation, the medial and lateral pontine gray, the parabrachial region, and the accessory inferior olive. This pattern of IGL and VLG projections is consistent with our understanding of the distinct functions of each of these ventral thalamic derivatives.     

Ultradian rhythmic neuronal oscillation in the intergeniculate leaflet

Our paper is the first to describe ultradian rhythmic neuronal oscillation in the intergeniculate leaflet (IGL) of the rat. We recorded a multiple-unit neuronal activity (MUA) from dorsal to ventral parts of the lateral geniculate nucleus (LGN) in anaesthetized rats. In all the subdivisions of the lateral geniculate complex we observed spontaneous irregular firing rates of cells. However only at the anatomical localisation of the IGL, after the light was on, those responses exhibited burst firing with a constant interburst interval, which lasted several hours until the light was off. The duration of that rhythmic oscillation obtained by means of Fourier's analysis was approximately 124 s. To date we have not had sufficient data to discuss possible mechanisms of this neuronal rhythmicity. We can only conclude that light is the most important stimulus not only for suprachiasmatic nuclei (SCN), but also for the IGL. On the other hand, we can neither exclude nor confirm that in order to evoke ultradian rhythmical oscillation in the IGL, in addition to light also non-photic information is necessary.     

The serotonergic inhibition of slowly bursting cells in the intergeniculate leaflet of the rat

Electrophysiological studies combined with local neurotoxic lesions were conducted on anaesthetized rats in order to determine whether the dorsal raphe nucleus (DRN) inhibits the intergeniculate leaflet (IGL) of the lateral geniculate nucleus by means of innervation by serotonin-containing fibres. In the control animals, electrical stimulation of the DRN induced the long-latency and long-lasting inhibition of the neuronal firing of the IGL cells that are characterized by rhythmic, slow-bursting activity in light conditions. The electrical destruction of the DRN resulted in an increase in the firing rate of the recorded IGL cells, whilst at the same time not affecting the rhythmic, bursting pattern of the activity. In the second group of animals, local neurotoxic lesion of serotonergic fibres was performed by injection of the toxin 5,7-dihydroxytryptamine into the IGL. After 10 days of postoperative recovery, electrophysiological experiments were performed on the toxin-treated rats. In these animals, electrical stimulation as well as electrical lesion of the DRN did not induce any change in the firing of the slowly bursting cells in the 5,7-dihydroxytryptamine-injected IGL. The results obtained provide evidence that inhibition of the IGL slowly bursting cells, by innervation from the dorsal raphe, is mediated by the release of serotonin. Furthermore, the observed serotonergic inhibition of the light-dependent activity of slowly bursting cells can contribute to the neuronal mechanism gating the information that flows through this nucleus to the vestibular, visuomotor, circadian and sleep/arousal systems, with which the IGL is strongly interconnected.     

Estrogen receptor beta (ERbeta) mRNA and protein in serotonin neurons of macaques.

This study used double in situ hybridization (ISH) to examine the colocalization of estrogen receptor beta (ERbeta) mRNA in serotonin neurons of rhesus macaques (Macaca mulatta). In addition, immunocytochemistry (ICC) was used to examine the expression and regulation of ERbeta protein in raphe neurons of the macaque midbrain. For double ISH, monkey specific riboprobes for ERbeta incorporating radiolabeled-UTP and a riboprobe for the human serotonin reuptake transporter (SERT) incorporating digoxigenin were applied to midbrain sections from spayed rhesus macaques. ERbeta mRNA hybridization signal was expressed in most cells containing SERT mRNA in the dorsal and median raphe and pons. There were also non-SERT neurons expressing ERbeta mRNA. In addition, ERbeta protein was detected with an affinity purified polyclonal antibody generated against a synthetic peptide corresponding to the D domain of human ERbeta conjugated to bovine serum albumin (provided by Dr. Philippa Saunders, MRC, Edinburgh). Midbrain sections containing the dorsal raphe from spayed rhesus macaques with and without hormone replacement therapy were processed for ERbeta immunostaining. ERbeta protein was detected at a similar intensity and in a similar number of cells in the dorsal raphe neurons in all treatment groups. Thus, the expression of ERbeta protein in the dorsal raphe was consistent with the expression of ERbeta mRNA. In conclusion, ERbeta mRNA is expressed by serotonin neurons and it is translated to protein. ERbeta protein, like ERbeta mRNA, is detected at similar levels in the presence or absence of ovarian hormones.     

Estrogen decreases hypothalamic angiotensin II AT1 receptor binding and mRNA in the female rat

Estrogen has been shown to modulate angiotensin II (AngII)-regulated behaviors, such as thirst, and may do so by influencing the central renin–angiotensin system (RAS). While numerous studies have attempted to correlate changes in AngII receptors or other components of the RAS with estrogen treatment, the low abundance of these genes has made comparisons difficult. Generally, such experiments have relied on traditional approaches to analyze gene expression that often restrict the experimenter to studying only a few mRNA species, whereas a behavior as complex as thirst may be influenced by changes in multiple genes. The present experiments utilized quantitative receptor autoradiography and mRNA expression profiling to identify and compare AngII receptors and their mRNA levels as well as other components of the RAS in female rat pituitary and hypothalamic–thalamic–septal (HTS) tissue samples. This relatively new approach to the study of gene expression permits the simultaneous comparison of multiple genes from a single tissue sample. These studies revealed that ovariectomized (OVX) female rats treated with estradiol benzoate (EB) had a 30%–40% reduction in the levels of AT₁ receptor mRNA in pituitary and HTS samples as compared to OVX, control animals. In the pituitary, the mRNA levels for angiotensinogen (AGT) were increased by 45% following estrogen administration. In addition, a reduction in [¹²⁵I]-AngII binding to AT₁ receptors in the pituitary and the subfornical organ was measured following estrogen treatment. These results suggest that estrogen may modulate the pituitary and central RAS through a coordinate regulation of the angiotensin receptors and the levels of newly synthesized AngII.     

Blockade of GABAA receptors disrupts isoperiodic neuronal oscillations in the intergeniculate leaflet of the rat

The intergeniculate leaflet of the thalamus is, besides the suprachiasmatic nucleus of the hypothalamus, the other important neuronal element of the mammalian biological clock. The extracellularly recorded activity of neurons constituting the intergeniculate leaflet, recorded in vivo, is characterized by distinct, very regular ultradian oscillations. The majority of neurons in the circadian timing system are GABAergic. Many, if not all, neurons of the suprachiasmatic nucleus and intergeniculate leaflet contain GABA. In the present study we examined the effects of the GABA(A) receptor antagonist bicuculline and the chloride channel blocker picrotoxin on isoperiodic neuronal oscillations in the intergeniculate leaflet of rats. We recorded extracellular multiple-unit neuronal activity from the intergeniculate leaflet of anesthetized rats. During the recording of isoperiodic oscillations, bicuculline or picrotoxin were stereotaxically injected at different concentrations into the lateral ventricle of rat brain. In all the experiments, injection of GABA(A) receptor antagonists transiently disrupted the isoperiodic phasic discharge recorded from the intergeniculate leaflet. These data suggest that GABA(A) receptors are involved in the generation of ultradian rhythmical neuronal oscillations in rat intergeniculate leaflet.     

Effects of intergeniculate leaflet lesions on circadian rhythms in the mouse.

We investigated effects of bilateral electrolytic intergeniculate leaflet (IGL) lesions (IGLX) on circadian rhythms of the wheel-running activity in mice kept under constant darkness conditions (DD). As a result of complete bilateral IGL lesions, the period of a free-running rhythm was lengthened by ca. 45 min in comparison with a pre-operational one. The present findings confirm results obtained previously with other animals. However, for the first time locomotor activity levels were precisely assessed in mice (the number of wheel revolutions/day) before and after IGL lesions. A computer analysis of data showed a considerable decrease in that activity (by 68% on an average) in mice with complete bilateral IGL lesions in comparison with IGL-non-lesioned individuals. The obtained results show that also in mice IGL constitutes an anatomically important element of the mechanism of a circadian time-keeping system, which mediates the transfer of non-photic information to the suprachiasmatic nuclei (SCN) by modulating their activity.     

Non-photic manipulations induce expression of Fos protein in the suprachiasmatic nucleus and intergeniculate leaflet in the rat

Expression of Fos protein in the suprachiasmatic nucleus (SCN) and intergeniculate leaflet (IGL) is considered a cellular correlate of light-induced phase-shift of circadian rhythms in rodents. Non-photic stimuli also induce phase shifts, but their effects on Fos expression have not been established. We examined induction of Fos protein in SCN and IGL regions, in response to cage change, intraperitoneal saline injection, and restraint stress. Fos immunoreactivity was observed in SCN and IGL regions, with greater expression observed in IGL during the light phase of the light-dark cycle. Results suggest that cells in SCN and IGL respond to several types of non-photic manipulations and that expression of Fos in these regions is not light-specific.     

Estrogen receptor beta immunoreactivity in differentiating cells of the developing rat cerebellum

Estrogen receptors (ER) play a significant role in the development of some regions of the mammalian brain. Recently, ER-beta (ERbeta) mRNA and protein were shown to be expressed in the rat cerebellum. In the present study, the ontogeny of ERbeta protein expression was examined in the rat cerebellum during postnatal development. Western blot analysis indicated that a single ERbeta-like immunoreactive species of approximately 55 kDa was present in protein lysates prepared from the cerebella of female and male Sprague-Dawley rat pups. Immunocytochemical analysis of cerebellar sections from the midline vermis revealed that during development, the expression of ERbeta varied with age and cell-type, but not sex. In the developing cerebellum, highest levels of ERbeta-immunoreactivity (IR) were detected in neurons during neurite growth, and in some glia during migration. Throughout the first postnatal week, ERbeta-IR was localized to differentiating granule cells in the external germinal layer and to migrating glia. Differentiating granule cells expressed detectable levels of ERbeta throughout development. In Purkinje cells, ERbeta-IR was first detected on postnatal day 6 (P6), with peak intensities of immunostaining coinciding with the initiation of axonal and dendritic growth that occurs between P7 and P8. Expression of ERbeta-IR remained high during maturation of Purkinje cell dendrites, and then decreased to a lower level maintained in the adult. From the third postnatal week, ERbeta-IR was also detected in the later developing Golgi, stellate, and basket neurons. These results suggest that ERbeta may play a role in growth-related mechanisms during differentiation of cerebellar neurons and glia.     

Distribution of substance P and neurokinin-1 receptor immunoreactivity in the suprachiasmatic nuclei and intergeniculate leaflet of hamster, mouse, and rat

The circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) receives photic information directly via the retinohypothalamic tract (RHT) and indirectly from retinally innervated cells in the thalamic intergeniculate leaflet (IGL) that project to the SCN. Using standard immunohistochemical methods, we examined the presence and distribution of substance P (SP) and the neurokinin-1 receptor (NK-1) in the SCN and IGL of rat and determined whether the patterns of immunostaining generalized to the SCN and IGL of Syrian hamster, Siberian hamster, and mouse. Terminals immunoreactive for SP were sparse within the SCN of Siberian and Syrian hamsters and mouse but were intense in the ventral, retinally innervated portion of the rat SCN. Immunostaining for the NK-1 receptor was mainly absent from the SCN of hamster and mouse. In contrast, a plexus of NK-1-ir cells and processes that was in close proximity to SP-ir terminals was found in the ventral SCN of the rat. Substance P-ir terminals were observed in the IGL of all four species, as were NK-1-ir cells and fibres. Double-labelled IGL sections of hamster or rat revealed SP-ir terminals in close apposition to NK-1-immunostained cells and/or fibres. These data indicate that SP could be a neurotransmitter of the RHT in rat, but not in hamster or in mouse, and they highlight potential species differences in the role of SP within the SCN circadian pacemaker. Such species differences do not appear to exist at the level of the IGL, where SP-ir and NK-1-ir were similar in all species studied.     

Orexin fibers form appositions with Fos expressing neuropeptide-Y cells in the grass rat intergeniculate leaflet

Neuropeptide-Y (NPY) cells in the intergeniculate leaflet (IGL) are known to modulate effects of arousal on the mammalian circadian system. However, the route through which this information reaches the IGL has not been established. Here, we provide evidence that the orexins (hypocretins) are uniquely positioned as a potential source of activity state feedback to the IGL in the grass rat, Arvicanthis niloticus. Specifically, many NPY cells in the grass rat IGL exhibit orexin-A (OXA) fiber appositions. Furthermore, NPY cells contacted by OXA fibers are significantly more likely to express Fos during nocturnal wheel running than are NPY cells without such contacts (P < 0.001).     

Estrogen and progesterone affect cocaine pharmacokinetics in female rats

Several studies have reported sex differences in behavioral responses to cocaine whereby females display a greater degree of locomotor activity. Fluctuations in estrogen and progesterone during the estrous cycle have been postulated to underlie these behavioral differences. In this study, we tested the hypothesis that hormonal replacement (estrogen or progesterone) in ovariectomized rats affects cocaine pharmacokinetics. We found that estrogen replacement did not affect cocaine-induced locomotor activity, but progesterone attenuated locomotor counts in comparison with control groups receiving only sesame oil. Estrogen, however, decreased brain levels of cocaine and norcocaine 30 min after cocaine administration in comparison to the group-receiving vehicle at that time point. In addition, in progesterone-treated rats, levels of benzoylecgonine and ecgonine methylester were higher at 30 min post-administration than at 15 min. No changes were found in blood levels of the metabolites. These findings suggest that while progesterone has an impact on locomotor behavior, pharmacokinetic effects may have a limited role in mediating behavioral responses to cocaine.     

Estrogen regulates GFAP-expression in specific subnuclei of the female rat interpeduncular nucleus: a potential role for estrogen receptor beta

We previously demonstrated that in rat, astrocytic glial fibrillary acidic protein- (GFAP) expression in the interpeduncular nucleus (IPN) was responsive to testosterone and in females the intensity of GFAP-immunoreactivity (IR) followed the periodic hormonal changes of the estrous cycle. The aim of this study was to test whether 17beta-estradiol (E(2)), in the absence of other ovarian hormones, can influence GFAP-expression within individual subnuclei of the IPN and to determine the cellular distribution of estrogen receptor beta (ERbeta) in the IPN. Quantitative surface-density analysis was used to compare the intensity of GFAP-IR at different anterio-posterior (AP) levels of the IPN in ovariectomized female rats 24 h after treatment with E(2) or vehicle. Estrogen-treatment resulted in a significant increase in GFAP-IR in the rostrolateral subnucleus of the IPN at AP: -5.60, in the lateral-, dorsolateral-, dorsomedial- and central subnuclei at -6.04 and in the lateral subnucleus at -6.72. No significant differences were observed at -5.80 and -6.30. These results indicate that E(2), in the absence of other ovarian hormones, modulates GFAP-expression within select IPN subnuclei and that these affects are dependent on position along the AP axis. To determine whether ERbeta was a possible mediator of the observed estrogenic effects, adjacent section pairs of the IPN were immunostained for ERbeta or GFAP. Using the 'mirror' method, ERbeta-IR was detected in the cytoplasm of GFAP-immunopositive astroglia and in the nuclei of GFAP-immunonegative neurons. These findings suggest that in the IPN, E(2) may directly modulate GFAP-expression through ERbeta-mediated mechanisms.     

Regulation of serotonin release in the Syrian hamster intergeniculate leaflet region

In mammals, the thalamic intergeniculate leaflet (IGL) conveys behavioral (non-photic) phase-resetting information to the circadian clock of the suprachiasmatic nucleus. Here we report a 24 h fluctuation in in vivo serotonin release in the hamster IGL region, peaking at night. Novel wheel exposure at midday, a stimulus that can reset circadian phase, activates the release of serotonin in the IGL region. In addition, electrical stimulation of the dorsal raphe nucleus acutely increases serotonin release the IGL region, confirming a functional serotonergic projection from this nucleus to the IGL. Collectively, these findings suggest that behavioral and/or circadian changes in dorsal raphe nuclear activity could modulate serotonin-mediated activities of the IGL.     

Estrogen receptor alpha and beta immunoreactive neurons in normal adult and aged female rat hippocampus: a qualitative and quantitative study

We have studied the distribution pattern and levels of expression of two estrogen receptor (ER) subtypes, ERalpha and ERbeta, in the normal adult (n = 10) and the aged (n = 10) female rat hippocampus with the objective to establish baseline data and the changes that occur during aging. Techniques including immunohistochemical localization, co-localization with double immunofluorescence and confocal microscopy, image analysis including neuronal counts/mm(2) area and measurements of optical density (OD) of immunoreactivity in immunoreactive neurons and Western blot analysis have been used. The results revealed ERalpha and ERbeta positive neurons in all subfields of the hippocampus with maximum presence in the stratum pyramidale of CA3. Some stained neurons in CA3 exhibited pyramidal neuron like morphological characteristics; such neurons were not found in CA1. All other immunoreactive neurons showed non-pyramidal neuron like morphological characteristics. Neuronal counts revealed a significant decrease in the number of immunoreactive neurons in CA3-CA1 of aged hippocampus. The percent decrease in counts of the immunoreactive neurons/mm(2) area in the aged rat (compared to the adult) was 78% for the ERalpha and 88% for the ERbeta (P < 0.001) in CA3. In CA1, it was 56% (P < 0.001) and 41% (P < 0.01) respectively. The OD of immunoreactivity was significantly decreased (P < 0.01) in CA3 but increased (P < 0.01) in the CA1 immunoreactive neurons. Western blot analysis also showed a significant decline (P < 0.01) in the levels of the ERalpha and ERbeta proteins in the aged hippocampus. Co-localization revealed that the two ER subtypes do co-exist in the same hippocampal neurons.     

Photoperiodic dependent changes in the number of neurons containing mRNA encoding neuropeptide Y in the intergeniculate leaflet of the Syrian hamster

The intergeniculate leaflet (IGL) is a distinct division of the lateral geniculate complex that participates in the regulation of the circadian rhythm through its projections to the circadian pacemaker located in the suprachiasmatic nuclei of the hypothalamus. A high number of neuropeptide Y (NPY) cell bodies has been described in the IGL by immunohistochemistry and in situ hybridization. The present study investigated whether NPY in the IGL is influenced by the length of the daily photoperiod. By using in situ hybridization we show a significant increase of the number of NPY mRNA containing neurons in the mid-part of the IGL of Syrian hamsters maintained in a short photoperiod compared to those kept in a long photoperiod. On the other hand, NPY mRNA expression per cell in the IGL is similar in both photoperiods tested.