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Endocrinology, doi:10.1210/en.2005-1262
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Endocrinology Vol. 147, No. 5 2197-2202
Copyright © 2006 by The Endocrine Society

Vasoactive Intestinal Polypeptide Regulates Dynamic Changes in Astrocyte Morphometry: Impact on Gonadotropin-Releasing Hormone Neurons

Lynnette M. Gerhold and Phyllis M. Wise

Department of Neurobiology, Physiology, and Behavior, College of Biological Sciences, University of California, Davis, Davis, California 95616

Address all correspondence and requests for reprints to: Lynnette Gerhold, Ph.D., Northwestern University, Department of Neurobiology and Physiology, 2205 Tech Drive, Evanston, Illinois 60208. E-mail: l-gerhold{at}northwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent studies suggest that astrocytes modulate the GnRH-induced LH surge. In particular, we have shown that the surface area of astrocytes that ensheath GnRH neurons exhibits diurnal rhythms. Vasoactive intestinal polypeptide (VIP) influences numerous aspects of astrocyte function in multiple brain regions and is a neurotransmitter in the suprachiasmatic nucleus (SCN) that affects GnRH neurons. The goals of this study were to: 1) assess whether astrocytes that surround GnRH neurons express VIP receptors, 2) determine the effects VIP suppression in the SCN on the morphometry of astrocytes surrounding GnRH neurons, and 3) assess whether this effect mimics aging-like changes in surface area of astrocytes. Young rats were ovariectomized (d 0), implanted with cannulae into the SCN (d 5), injected with VIP antisense (antioligo) or random sequence oligonucleotides, implanted with capsules containing 17ß-estradiol dissolved in oil (d 7), and perfused at 0300, 1400, and 1800 h (d 9). Brains were processed for immunocytochemistry. Our results demonstrate that astrocytes in close apposition to GnRH neurons express VIP receptors. Antioligo treatment blocked diurnal rhythms in surface area of astrocytes ensheathing GnRH neurons. The absence of diurnal rhythms resembles observations in middle-aged rats. Together these findings suggest that the ability of the VIP-containing neurons in the SCN to relay diurnal information to GnRH neurons may be by influencing dynamic changes in the morphometry of astrocytes that surround GnRH neurons. Furthermore, the absence of a VIP rhythm in aging animals may lead to altered GnRH activity via astrocyte-dependent mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING THE PAST several years, it has become clear that astrocytes modulate neuron-neuron communication based on evidence that astrocytes express ion channels, neurotransmitters, and hormone receptors; modulate release of neurotransmitters by neurons; and influence neurite outgrowth and neuronal survival after a variety of insults (1, 2, 3, 4, 5, 6, 7). More recently we have come to appreciate that astrocytes in different brain regions behave in a region-specific manner as a result of the biochemical microenvironment determined by hormones and neurotransmitter release in that brain region (8, 9, 10, 11, 12, 13). In particular, estradiol exerts different effects on the morphometry of astrocytes in different regions of the hypothalamus and hippocampus. For instance, estradiol induces opposing morphological and functional changes in astrocytes that are intimately located near GnRH neuronal cell bodies, compared with GnRH nerve terminals (9, 10, 13, 14, 15, 16).

Studies from our laboratory (16) demonstrate that the surface area of astrocytes that surround GnRH neuronal cell bodies in the organun vasculosum of the lamina terminalis (OVLT) and the rostral region of the medial preoptic nucleus (rMPN) exhibit diurnal rhythms: surface area is high on the morning of proestrus and decreases throughout the afternoon. This retraction of processes and reduction in surface area is thought to allow stimulatory inputs to GnRH neurons to induce preovulatory GnRH synthesis and secretion. Vasoactive intestinal polypeptide (VIP) is a neurotransmitter and neurotrophic factor (17, 18, 19) that exerts broad and diverse actions in several brain regions by acting on neurons and astrocytes to stimulate the release of one or more factors that directly or indirectly affect synaptogenesis of neurons (17, 19). In the suprachiasmatic nucleus (SCN) within the hypothalamus, VIP entrains diurnal rhythms in the SCN and GnRH neurons (20); whereas, in the cortex, VIP acts as a neurotrophic factor. Originally, VIP was thought to act predominantly by directly modulating neuronal activity. More recently it has become clear that VIP influences neurons in the cortex through activation of VIP receptors located on astrocytes (17, 19). However, it is not known whether and/or how VIP influences diurnal rhythms in the morphometry of astrocytes that surround GnRH neurons.

Aging involves changes in GnRH neuronal activation (21, 22, 23), and VIP is critical to the activation of GnRH neurons (20). In the SCN, VIP mRNA decreases with age (24). Aging also attenuates dynamic changes in astrocyte morphometry (16). This age-related attenuation in diurnal rhythms of VIP and astrocyte morphometry has been correlated with diminution of diurnal rhythms in GnRH neuronal synthesis and activity (21, 22, 23, 25) and an attenuated and delayed LH surge (26).

The goal of this study was to assess whether there is a functional relationship between the diurnal rhythmicity in VIP activity in the SCN and the rhythmicity in astrocyte morphometry. We hypothesized that VIP may indirectly affect GnRH neurons by influencing astrocytes that are associated with these neurons. Furthermore, we tested the hypothesis that blockade of VIP rhythms would result in aging-like changes in the diurnal rhythm of the morphometry of astrocytes surrounding GnRH neurons, which in turn may lead to a decline in GnRH neuronal function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Young (2–3 months old) and middle-aged (9–12 months old) female Sprague Dawley rats (Zivic-Miller; Penelope, PA) were maintained on a 14-h light, 10-h dark cycle (lights on at 0400 h) with food and water available ad libitum. Estrous cyclicity was monitored by daily vaginal lavage for at least 3 wk before use. Only young rats that exhibited at least two consecutive 4-d estrous cycles and middle-aged rats that exhibited estrous cycles of regular or irregular length (5–8 d) were used. All rats were ovariectomized (OVX; d 0) and implanted sc with a SILASTIC brand capsule (0900 h, d 7; Dow Corning, Midland, MI; supplied by Konigsberg Instruments, Pasadena, CA) containing either sesame oil or 17ß-estradiol (E2; Sigma Chemical Co., St. Louis, MO) dissolved in sesame oil (180 µg/ml; young: 30 mm capsule; middle-aged: 40 mm capsule; 0.062 in./0.125 in., inner/outer diameter). This E2 treatment paradigm produces estradiol levels around 15 pg/ml by d 2 after implantation, and LH surges occur between 1300 and 1900 h, with peak concentrations at approximately 1500–1600 h (27). The OVLT/rMPN contains the subpopulation of GnRH neurons that regulate LH secretion (28, 29). We examined these areas based on previous work showing that VIP predominantly innervates the GnRH neurons in this region and not GnRH neurons that are lateral to these regions (23, 30).

Stereotaxic surgery
Stereotaxic surgeries in young animals were performed based on the protocols of Harney et al. (31) and Gerhold et al. (20), which are briefly described below. Five days after OVX (d 5), rats were anesthetized with ketamine (49 mg/ml)/xylazine (1.8 mg/ml) and implanted with bilateral guide tubes directed stereotaxically at the SCN (1.5 mm apart; 9 mm in length; 26 gauge; Plastics One Inc., Roanoke, VA). Two days after bilateral guide tube implantation (d 7), VIP antisense or random sequence oligos (0.5 µg in 0.5 µl saline per side; Invitrogen Life Technologies, Carlsbad, CA) were infused over a 4-min interval at 0800 h through cannulae implanted bilaterally in the guide tubes. The antisense oligonucleotides were 20-mers complementary to the cap site (5'-GCTCTGCACTACAACCTGAC-3') and translation start site (5'-TTGCTTCTGGATTCCATCTC-3') of the rat VIP mRNA (32). Control oligonucleotides had the same ATGC content as antisense oligonucleotides but in random order that had no significant homology to any known peptide localized in the SCN. At the same time, rats were implanted with SILASTIC brand capsules containing E2 dissolved in sesame oil. Rats were killed 2 d later (d 9) at 43 h (0400 h), 53 h (1400 h), and 57 h (1800 h) after injection. Antisense and random sequence oligo-treated rats as well as a group of middle-aged regularly cycling female rats (OVX, d 0, E2 implanted d 7, and killed d 9) were transcardially perfused with 4% paraformaldehyde (Sigma), and the brains were removed, sectioned coronally, and stored in cyroprotectant at –20 C until processed for immunocytochemistry. The SCN was checked for correct cannulae placement as the brains were sectioned (n = 6–10 rats per time point per treatment). VIP antisense treatment effectively suppresses VIP concentrations in the SCN (20).

Immunocytochemistry: VPAC2 receptor/glial fibrillary acidic protein (GFAP)
Coronal brain sections (40 µm) were cut in a cryostat (Microm, Kalamazoo, MI) starting at the level of the medial diagonal band of Broca (bregma 0.20 mm) (33) through the arcuate nucleus (bregma –3.80 mm) (33). Each brain was collected in a series of six sets with every sixth section represented in a group and stored in cyroprotectant until processed for immunocytochemistry. Antibodies against GFAP (Sigma) and VPAC2 receptor (VIP receptor subtype) (Santa Cruz Biotechnology, Santa Cruz, CA) were used to determine whether astrocytes express this VIP receptor subtype. On d 1, sections from one series that contains every sixth section were rinsed in 0.1 M Tris-buffered saline (TBS) and blocked with 10% normal horse serum plus 0.4% Triton X-100 (NHS-X) for 1 h at room temperature. Next, sections were incubated in mouse anti-GFAP (1:50,000) in 2% NHS-X overnight at 4 C. On d 2, sections were rinsed in TBS and incubated in Cy2-conjugated antimouse IgG (Jackson ImmunoReseach Laboratories, West Grove, PA) diluted 1:500 in 2% NHS-X for 1 h at room temperature. Sections were rinsed in TBS and incubated in goat anti-VPAC2 receptor (1:1000) in 2% NHS-X for 2 d at 4 C. On d 4, sections were rinsed in TBS and incubated in Cy3-conjugated antigoat IgG (Jackson ImmunoReseach Laboratories) diluted 1:500 in 2% NHS-X for 1 h at room temperature. Sections were rinsed and mounted on slides and coverslips were applied using Permount (Fisher Scientific, Pittsburgh, PA). For a control experiment, a separate set of sections was processed for GFAP immunoreactivity; however, the primary antibody, VPAC2, was omitted from the 2% NHS-X, whereas incubating for 2 d before incubating with the secondary Cy3-conjugated antigoat IgG for 1 h. No VPAC2 receptor immunoreactivity was detected with omission of the antibody. In addition, we used the blocking peptide supplied (Santa Cruz Biotechnology) with this antibody and added it to the antibody to determine specificity of the antibody. No VPAC2 receptor immunoreactivity was detected.

Immunocytochemistry: GFAP/GnRH
Antibodies against GnRH (LR-5, Benoit, Montreal, Canada) and GFAP (Sigma) were used to identify astrocytes surrounding GnRH neurons. Identifying astrocytes by using the structural protein GFAP accounts for only a percentage of the total astrocyte cell body and processes. However, small changes in this structural protein may lead to correlative changes in the astrocyte processes. On d 1, sections from one series that contains every sixth section were rinsed in TBS and blocked with 10% NHS-X for 1 h at room temperature. Next, sections were incubated in mouse anti-GFAP (1:50,000) in 2% NHS-X overnight at 4 C. On d 2, sections were rinsed in TBS, incubated in biotinylated antimouse IgG diluted 1:500 in 2% NHS-X for 1 h at room temperature, followed by incubation in avidin-biotin complex (Vectastain kit, Vector Laboratories, Burlingame, CA) in TBS plus 0.4% Triton X-100 for 1 h at room temperature. Finally, sections were incubated in 0.025% diaminobenzidine (DAB; Sigma) with 0.02% ammonium nickel sulfate (Fisher Scientific) and 0.1 µl/ml of 30% hydrogen peroxide in TBS for 10 min at room temperature. After the DAB reaction, sections were rinsed in TBS and then incubated in rabbit anti-GnRH (1:100,000) in 2% NHS-X overnight at 4 C. On d 3, sections were rinsed in TBS, incubated in biotinylated antirabbit IgG diluted 1:500 in 2% NHS-X for 1 h at room temperature, followed by incubation in avidin-biotin complex (Vectastain kit, Vector Laboratories) in TBS plus 0.4% Triton X-100 for 1 h at room temperature. Finally, sections were incubated in 0.025% DAB (Sigma) with 0.1 µl/ml of 30% hydrogen peroxide in TBS for 10 min at room temperature. Each step was followed by rinses in TBS (3 x 5 min). Sections were mounted on slides and coverslips were applied using Permount.

Measuring astrocyte surface area
Bright-field photomicrographs using a x40 objective were taken of astrocytes that surround GnRH neurons using a Leica DC 500 camera attached to a Leica DMLB microscope (JH Technologies, San Jose, CA). Photomicrographs were analyzed using Metamorph Image Analysis software 6.1 (Universal Imaging Corp., Downing, PA). Astrocyte surface area was obtained by using the tracing tool to outline the astrocyte and all processes, and Metamorph was calibrated to determine the area in the outlined region. Only astrocytes that were in close apposition to GnRH neurons were measured (two astrocytes each per five GnRH neurons in the OVLT and two astrocytes each per 10 GnRH neurons in the rMPN).

Statistical analysis
Two-way ANOVA followed by Bonferroni’s post hoc test were used to analyze surface area significance of astrocytes that surround GnRH in antisense and random sequence oligo-treated rats and middle-aged rats for effects of treatment, time, and treatment x time interactions. All statistics were performed using Prism 4.0 software (GraphPad, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VPAC2 receptor expression
Brain sections were double labeled with GFAP, a marker for astrocytes and the VIP receptor subtype, VPAC2, to assess whether astrocytes express the same receptor for VIP that GnRH neurons and neurons in the SCN express. Astrocytes in the rMPN and OVLT express the VIP receptor subtype, VPAC2 (Fig. 1Go) in OVX rats treated with E2. VPAC2 receptors were primarily localized in the processes and also the soma of astrocytes (Fig. 1Go). VPAC2 receptor immunoreactivity (IR) was also seen in cells that are adjacent to astrocytes (data not shown).


Figure 1
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  		FIG. 1. Photomicrograph of astrocytes expressing VPAC2 receptors in the OVLT. A, VPAC2 receptor immunoreactive cell stained with Cy3 (red). B, GFAP-IR cell stained with Cy2 (green). C, Overlay of VPAC2 receptor immunoreactive cell colocalized with a GFAP immunoreactive cell.

 
Effects of VIP suppression in the SCN on astrocytes in the OVLT and rMPN
In the OVLT of rats that were injected with random sequence oligos into the SCN, the surface area of astrocytes that surround GnRH neurons exhibited a diurnal rhythm (Fig. 2AGo) as we have previously observed in noncannulated proestrous rats (16). Astrocyte surface area in random sequence oligo-treated animals was high at 0300 h and decreased significantly by 1400 h (P < 0.01). In contrast, rats treated with antisense oligos did not exhibit a diurnal rhythm in astrocyte surface area (Fig. 2AGo) in the OVLT; antisense oligo treatment prevented the decline in astrocyte surface area at 1400 h.


Figure 2
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  		FIG. 2. The surface area of astrocytes that surround GnRH neurons in the OVLT (A) and rMPN (B) of rats treated with random sequence oligos throughout the day exhibited a diurnal rhythm. Treatment with antisense oligos in the SCN disrupted the diurnal rhythm in the OVLT and rMPN. Asterisks indicate levels of significance (*, P < 0.05; **, P < 0.01) between time points. Pound signs indicate levels of significance (#, P < 0.05; ##, P < 0.01; ###, P < 0.001) between treatments.

 
In the rMPN, the surface area of astrocytes that surround GnRH neurons in random sequence oligo-treated rats also displayed a diurnal rhythm (Fig. 2BGo). Surface area of astrocytes in the rMPN was similar to that observed in the OVLT, in that the surface area was high at 0300 h and was significantly decreased at 1400 h (P < 0.05). Antisense oligo treatment effectively blocked the decrease in surface area of astrocytes that surround GnRH neurons at 1400 h (Fig. 2BGo).

Aging affects the surface area of astrocytes in the OVLT and rMPN
In the OVLT of middle-aged rats, the surface area of astrocytes that surround GnRH neurons no longer demonstrates a diurnal rhythm (Fig. 3AGo). The astrocyte surface area did not decline at 1400 or 1800 h in these rats, and the area of astrocytes seen in middle-aged animals across the day resembled the surface area of astrocytes measured in animals treated with antisense oligos (Fig. 2AGo). This lack of diurnal rhythms in the surface area of astrocytes that surround GnRH neurons is also seen in the rMPN of middle-aged rats, in that no decline in astrocyte surface area was seen at 1400 or 1800 h (Fig. 3BGo). The surface area of astrocytes in the rMPN of middle-aged rats was similar to the surface area of astrocytes observed in the rMPN of antisense oligo-treated rats (Fig. 2BGo).


Figure 3
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  		FIG. 3. GnRH. The surface area of astrocytes that surround GnRH neurons in the OVLT (A) and rMPN (B) in middle-aged (MA) rats did not exhibit a diurnal rhythm and was not different from the antisense oligo-treated rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
An accumulating body of evidence clearly establishes that dynamic bidirectional interactions between neurons and astrocytes are essential during development and adulthood to maintain normal brain function. These neuron-astrocyte interactions play important roles in the ability of the nervous system to respond to changing internal and external environments. Our studies expand on this growing understanding and suggest that VIP may act on astrocytes to regulate GnRH neurons in the hypothalamus. In OVX+E2-treated rats, in which diurnal LH surges occur at a predictable time every afternoon, we demonstrate that astrocytes identified by GFAP, which surround the GnRH neurons in the OVLT and rMPN: 1) exhibit a diurnal rhythm in surface area, 2) express receptors for VIP, and 3) lose the diurnal rhythm of surface area with age. Furthermore, we show that blockade of the diurnal rhythm in VIP expression in the SCN suppresses the diurnal rhythm in surface area of astrocytes surrounding GnRH neurons and that the lack of rhythmicity resembles the lack of rhythmic changes in astrocyte surface area in middle-aged rats.

The SCN contains an extensive network of astrocytes (34), and neurons in this brain region exhibit more coverage by astrocytes than neurons in the anterior hypothalamic areas (35). In the SCN, astrocyte processes are found between neuronal cell bodies and adjacent to presynaptic and postsynaptic sites (36). Astrocyte processes ensheath VIP and vasopressin neurons, with a greater percent of VIP neurons ensheathed by astrocytes (36). Treatment with agents that prevent glial metabolism or block astrocyte gap junction formation in the SCN disrupts circadian rhythms in neuronal activity, suggesting that astrocytes play a crucial role in maintaining circadian rhythms (37). However, conflicting results have been reported on whether diurnal rhythms in astrocyte morphology exist (35, 36, 38).

Astrocytes have been shown to play a role in the regulation of GnRH neurons and secretion. Astrocytes in close apposition to GnRH neurons, which may influence GnRH neuronal activation and release, display region-specific characteristics that are regulated differentially by circulating estradiol levels (10, 15). Astrocytes in the arcuate nucleus along with tanycytes that send processes to the median eminence increase their ensheathment of GnRH terminals on the afternoon of proestrus when estradiol levels are elevated, which limits inhibitory inputs that suppress GnRH release into the median eminence (13, 14). In contrast, our laboratory has demonstrated that astrocytes in the rMPN and OVLT, which regulate synaptic input to the GnRH neuronal cell bodies, exhibit a different diurnal pattern. During the afternoon of proestrus, astrocytes decrease their GFAP immunoreactivity, and this may lead to a decrease in the degree of ensheathment of GnRH neurons, which may lead to an increase in stimulatory inputs that induce GnRH synthesis and release (16) and thereby may limit inputs that may influence GnRH neurons.

Astrocyte-neuron interactions are bidirectional. Araque et al. (39) proposed the tripartite synapse consisting of the presynaptic and postsynaptic neurons along with astrocyte processes that can either physically modify synaptic communication or chemically alter synapse firing due to the close proximity of astrocytes around synapses. In the current study, we observed VPAC2 receptor-IR in GFAP-IR cells in the OVLT and rMPN as well as VPAC2 receptor-IR colocalized with cells with neuronal-like morphology (data not shown) that were in close apposition to GFAP-IR astrocytes. Our demonstration of VPAC2 receptors on astrocytes and neuron-like cells in conjunction with previous observations of the presence of VPAC2 receptors on GnRH neurons (40) suggest that VIP may influence GnRH neurons in numerous ways by affecting GnRH neurons directly, other neuronal phenotypes that communicate with GnRH neurons, and astrocytes that are in close proximity to GnRH or other neurons. We explored the possibility that one mechanism by which VIP affects GnRH neuronal activity is by modulating the surface area of astrocytes that surround GnRH neurons, which thereby influences the density of synapses on GnRH neurons.

Our studies demonstrate that VIP decreases astrocyte surface area around GnRH neurons at the time of the LH surge and could therefore indirectly enhance stimulatory inputs to synapse on GnRH neurons. One of these stimulatory inputs is VIP (30, 40). Work from our laboratory (20) demonstrated a stimulatory role of VIP from the SCN in inducing GnRH neuronal activation. One possibility is that VIP may also be able to influence GnRH neurons to induce changes in astrocyte morphology. Another example is estrogen receptor-containing neurons located in the AVPV (41), which project to the OVLT near GnRH neurons (42). This pathway may explain how estrogen’s stimulatory effects on the LH surge are communicated to GnRH neurons. Furthermore, some estrogen receptor-containing neurons in the AVPV have recently been shown to also contain {gamma}-aminobutyric acid and glutamate vesicles, in which an increase in glutamate-containing vesicles and a decrease in {gamma}-aminobutyric acid -containing vesicles occur at the time of the LH surge (43) and therefore potentially stimulate GnRH neurons.

It is interesting to note that Cashion et al. (16) did not see a decrease in astrocyte surface area on the afternoon of estrus when estradiol concentrations are low, even though VIP concentrations in the SCN are elevated at this time (44). It is possible that an increase in estradiol and the diurnal increase in VIP are both necessary to induce plastic changes in the surface area of astrocytes that surround GnRH neurons. In our studies, rats were implanted with E2-containing capsules to elucidate whether a daily signal is necessary to induce changes in astrocyte morphology. Indeed, blockade of VIP in the SCN prevented the afternoon decline in the surface area of astrocytes that surround GnRH neurons in E2-primed rats.

Aging dampens SCN VIP rhythmicity, which in turn decreases the diurnal plasticity of astrocytes. Cashion et al. (16), in agreement with our data, demonstrate that middle-aged rats no longer exhibit diurnal changes in the surface area of astrocytes that surround GnRH neurons. When we blocked the VIP rhythm and suppressed its concentrations in the SCN using antisense oligo methodology, we inhibited the diurnal changes in the surface area of astrocytes that are closely apposed to GnRH neuronal cell bodies. The absence of the astrocytes morphometry rhythm in our VIP antisense-treated rats is strikingly similar to what we observe in middle-aged rats that experience attenuated GnRH neuronal activation (20). Therefore, we conclude that VIP is necessary to induce cyclic changes in astrocyte morphometry and that the age-related loss of VIP in the SCN leads to a loss in the diurnal rhythm in astrocyte surface area.

In summary, VIP from the SCN plays a crucial role in regulating GnRH neuronal activity by directly influencing GnRH neurons (20, 30, 31, 40) and indirectly modulating the surface area of astrocytes that surround GnRH neurons, which allows for stimulatory inputs to GnRH neurons to occur at the right time to induce an LH surge. Furthermore, maintenance of VIP’s daily rhythm is of great importance to the synchronization of the SCN with GnRH neurons and astrocytes that surround GnRH neurons. Subtle changes in the ability of the biological clock to drive reproductive rhythms in middle-aged rats may underlie the transition to acyclicity and decline in reproductive function.


   Footnotes
 
This work was supported by National Institutes of Health Grants AG02224, AG17164 (to P.M.W.), and NS47875 (to L.M.G.).

Disclosure: L.M.G. and P.M.W. have nothing to declare.

First Published Online February 9, 2006

Abbreviations: DAB, Diaminobenzidine; E2, 17ß-estradiol; GFAP, glial fibrillary acidic protein; IR, immunoreactivity; NHS-X, normal horse serum plus Triton X-100; OVLT, organun vasculosum of the lamina terminalis; OVX, ovariectomized; rMPN, rostral region of the medial preoptic nucleus; SCN, suprachiasmatic nucleus; TBS, Tris-buffered saline; VIP, vasoactive intestinal polypeptide; VPAC2, VIP receptor subtype.

Received October 4, 2005.

Accepted for publication February 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Vernadakis A 1996 Glia-neuron interactions and synaptic plasticity. Prog Neurobiol 49:185–214[CrossRef][Medline]
  2. Araque A, Carmignoto, Haydon PG 2001 Dynamic signaling between astrocytes and neurons. Annu Rev Physiol 63:795–813[CrossRef][Medline]
  3. Hertz L, Zielke HR 2004 Astrocytic control of glutamatergic activity: astrocytes as stars of the show. Trends Neurosci 27:735–743[CrossRef][Medline]
  4. Ullian EM, Christopherson KS, Barres BA 2004 Role of glia in synaptogenesis. Glia 47:209–216[CrossRef][Medline]
  5. Trendelendburg G, Dimagl U 2005 Neuroprotective role of astrocytes in cerebral ischemia: focus on ischemic preconditioning. Glia 50:307–320[CrossRef][Medline]
  6. Perea G, Araque A 2005 Synaptic regulation of the astrocyte calcium signal. J Neural Transm 112:127–135[CrossRef][Medline]
  7. Garcia-Segura LM, Naftolin F, Hutchinson JB, Azcoitia I, Chowen JA 1999 Role astroglia in estrogen regulation of synaptic plasticity and brain repair. J Neurobiol 40:574–584[CrossRef][Medline]
  8. Ojeda SR, Dissen GA, Junier MP 1992 Neurotrophic factors and female sexual development. Front Neuroendocrinol 13:120–162[Medline]
  9. Garcia-Segura LM, Luquin S, Parducz A, Naftolin F 1994 Gonadal hormone regulation of glial fibrillary acidic protein immunoreactivity and glial ultrastructure in the rat neuroendocrine hypothalamus. Glia 10:59–69[CrossRef][Medline]
  10. Mong JA, Glaser E, McCarthy MM 1999 Gonadal steroids promote glial differentiation and alter neuronal morphology in the developing hypothalamus in a regionally specific manner. J Neurosci 19:1464–1472[Abstract/Free Full Text]
  11. Ojeda SR, Prevot V, Heger S, Lomniczi A, Dziedzic B, Mungenast A 2003 Glia-to-neuron signaling and the neuroendocrine control of female puberty. Ann Med 35:244–255[CrossRef][Medline]
  12. McCarthy MM, Todd BJ, Amateau SK 2003 Estradiol modulation of astrocytes and the establishment of sex differences in the brain. Ann NY Acad Sci 1007:283–297[CrossRef][Medline]
  13. Garcia-Segura LM, McCarthy MM 2004 Minireview: role of glia in neurodocrine function. Endocrinology 145:1082–1086[Abstract/Free Full Text]
  14. Mong JA, McCarthy MM 1999 Steroid-induced developmental plasticity in hypothalamic astrocytes: implications for synaptic patterning. J Neurobiol 40:602–619[CrossRef][Medline]
  15. McCarthy MM, Amateau SK, Mong JA 2002 Steroid modulation of astrocytes in the neonatal brain: implications for adult reproductive function. Biol Reprod 67:691–698[Abstract/Free Full Text]
  16. Cashion AB, Smith MJ, Wise PM 2003 The morphometry of astrocytes in the rostral preoptic area exhibits a diurnal rhythm on proestrus: relationship to the luteinizing hormone surge and effects of age. Endocrinology 144:274–280[Abstract/Free Full Text]
  17. Brenneman DE, Neale EA, Foster GA, d’Autremont SW, Westbrook GL 1987 Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide. J Cell Biol 104:1603–1610[Abstract/Free Full Text]
  18. Brenneman DE, Nicol T, Warren D, Bowers LM 1990 Vasoactive intestinal peptide: a neurotrophic releasing agent and an astroglial mitogen. J Neurosci Res 25:386–394[CrossRef][Medline]
  19. Brenneman DE, Phillips TM, Festoff BW, Gozes I 1997 Identity of neurotrophic molecules released from astroglia by vasoactive intestinal peptide. Ann NY Acad Sci 814:167–173[CrossRef][Medline]
  20. Gerhold LM, Rosewell KL, Wise PM 2005 Suppression of vasoactive intestinal polypeptide in the suprachiasmatic nucleus leads to aging-like alterations in cAMP rhythms and activation of gonadotropin-releasing hormone neurons. J Neurosci 25:62–67[Abstract/Free Full Text]
  21. Lloyd JM, Hoffman GE, Wise PM 1994 Decline in immediate early gene expression in gonadotropin-releasing hormone neurons during proestrus in regularly cycling, middle-aged rats. Endocrinology 134:1800–1805[Abstract/Free Full Text]
  22. Le WW, Wise PM, Murphy AZ, Coolen LM, Hoffman GE 2001 Parallel declines in Fos activation of the medial anteroventral periventricular nucleus and LHRH neurons in middle-aged rats. Endocrinology 142:4976–4982[Abstract/Free Full Text]
  23. Krajnak KM, Rosewell KL, Wise PM 2001 Fos-induction in gonadotropin-releasing hormone neurons receiving vasoactive intestinal polypeptide innervation is reduced in middle-aged female rats. Biol Reprod 64:1160–1164[Abstract/Free Full Text]
  24. Krajnak K, Kashon ML, Rosewell KL, Wise PM 1998 Aging alters the rhythmic expression of vasoactive intestinal polypeptide mRNA but not arginine vasopressin mRNA in the suprachiasmatic nuclei of female rats. J Neurosci 18:4767–4774[Abstract/Free Full Text]
  25. Rubin BS, Lee CE, Ohtomo M, King JC 1997 Luteinizing hormone-releasing hormone gene expression differs in young and middle-aged females on the day of a steroid-induced LH surge. Brain Res 770:267–276[CrossRef][Medline]
  26. Wise PM 1982 Alterations in proestrous LH, FSH, and prolactin surges in middle-aged rats. Proc Soc Exp Biol Med 169:348–354[CrossRef][Medline]
  27. Wise PM, Camp-Grossman P, Barraclough CA 1981 Effects of estradiol and progesterone on plasma gonadotropins, prolactin, and LHRH in specific brain areas of ovariectomized rats. Biol Reprod 24:820–830[Abstract]
  28. Petersen SL, McCrone S, Keller M, Shores S 1995 Effects of estrogen and progesterone on luteinizing hormone-releasing hormone messenger ribonucleic acid levels: consideration of temporal and neuroanatomical variables. Endocrinology 136:3604–3610[Abstract]
  29. Swanson LW 1987 The hypothalamus. In: Bjorklund A, Hokfelt T, eds. Handbook of chemical neuroanatomy. Amsterdam: Elsevier; 1–124
  30. van der Beek EM, Horvath TL, Weigant VM, van den Hurk R, Buijs RM 1997 Evidence for a direct neuronal pathway from the suprachiasmatic nucleus to the gonadotropin-releasing hormone system: combined tracing and light and electron microscopic immunocytochemical studies. J Comp Neurol 384:569–579[CrossRef][Medline]
  31. Harney JP, Scarborough K, Rosewell KL, Wise PM 1996 In vivo antisense antagonism of vasoactive intestinal peptide in the suprachiasmatic nuclei causes aging-like changes in the estradiol-induced luteinizing hormone and prolactin surges. Endocrinology 137:3696–3701[Abstract]
  32. Giladi E, Shani Y, Gozes I 1990 The complete structure of the rat VIP gene. Mol Brain Res 7:261–267[Medline]
  33. Paxinos G, Watson C 1997 The rat brain in stereotaxic coordinates. New York: Academic Press
  34. Morin LP, Johnson RF, Moore RY 1989 Two brain nuclei controlling circadian rhythms are identified by GFAP immunoreactivity in hamsters and rats. Neurosci Lett 99:55–60[CrossRef][Medline]
  35. Elliott AS, Nunez AA 1994 An ultrastructural study of somal appositions in the suprachiasmatic nucleus and anterior hypothalamus of the rat. Brain Res 662:278–282[CrossRef][Medline]
  36. Tamada Y, Tanaka M, Munekawa K, Hayashi S, Okamura H, Kubo T, Hisa Y, Ibata Y 1997 Neuron-glia interaction in the suprachiasmatic nucleus: a double labeling light and electron microscopic immunocytochemical study in the rat. Brain Res Bull 45:281–287
  37. Prosser RA, Edgar DM, Heller HC, Miller JD 1994 A possible glial role in the mammalian circadian clock. Brain Res 643:296–301[CrossRef][Medline]
  38. Lavialle M, Serviere J 1993 Circadian fluctuations in GFAP distribution in the Syrian hamster suprachiasmatic nucleus. Neuroreport 4:1243–1246[Medline]
  39. Araque A, Parpura V, Sanzgiri RP, Haydon PG 1999 Tripartite synapses: glia, the unacknowledged partner. Trends Neurosci 22:208–215[CrossRef][Medline]
  40. Smith MJ, Jennes L, Wise PM 2000 Localization of the VIP2 receptor protein on GnRH neurons in the female rat. Endocrinology 141:4317–4320[Abstract/Free Full Text]
  41. Simerly RB, Chang C, Muramatsu M, Swanson LW 1990 Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. J Comp Neurol 294:76–95[CrossRef][Medline]
  42. Gu GB, Simerly RB 1997 Projections of the sexually dimorphic anteroventral periventricular nucleus in the female rats. J Comp Neurol 384:142–164[CrossRef][Medline]
  43. Ottem EN, Godwin JG, Krishnan S, Petersen SL 2004 Dual-phenotype GABA/glutamate neurons in adult preoptic area: sexual dimorphism and function. J Neurosci 24:8097–8105[Abstract/Free Full Text]
  44. Krajnak K, Kashon ML, Rosewell KL, Wise PM 1998 Sex differences in the daily rhythm of vasoactive intestinal polypeptide but not arginine vasopressin messenger ribonucleic acid in the suprachiasmatic nuclei. Endocrinology 139:4189–4196[Abstract/Free Full Text]



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