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J Physiol (2003), 551.3, pp. 815-823
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.042739
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ABSTRACT |
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Chronic dehydration induces structural changes in the hypothalamic supraoptic nucleus (SON), including increased glutamate synapses and retraction of astroglial processes. We performed whole-cell recordings in acute hypothalamic slices to determine whether these changes increase tonic activation of presynaptic metabotropic glutamate receptors (mGluRs) by increasing ambient glutamate in the SON. Activation of presynaptic group III mGluRs caused a decrease in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in SON neurones that was significantly attenuated in slices from dehydrated rats (-27.8 %) compared with untreated rats (-41.7 %), suggesting a higher basal occupancy of mGluRs by ambient glutamate during dehydration. Blocking group III mGluRs caused an increase in the frequency of mEPSCs that was significantly higher in slices from dehydrated rats (+42.8 %) than untreated rats (+31.4 %), suggesting greater tonic activation of presynaptic mGluRs by ambient glutamate during dehydration. Increasing ambient glutamate levels by inhibiting astrocyte glutamate uptake resulted in a decrease in mEPSC frequency due to increased activation of presynaptic mGluRs. This was attenuated in slices from dehydrated rats (-35.4 %) compared with slices from untreated rats (-48.8 %), suggesting diminished astrocytic glutamate uptake during dehydration. Immunochemical analyses revealed a robust expression of the GLT-1 transporter protein in the SON, which was diminished in SON punches from dehydrated rats compared with untreated controls. Thus, dehydration leads to increased tonic activation of presynaptic mGluRs on glutamate terminals, consistent with a decrease in glutamate buffering capacity. The resulting reduction in glutamate release probability may compensate for the increase in glutamate release sites that occurs during dehydration.
(Resubmitted 10 March 2003; accepted after revision 12 June 2003; first published online 24 June 2003)
Corresponding author J. Tasker: Department of Cell and Molecular Biology, 2000 Percival Stern Hall, Tulane University, New Orleans, LA 70118-5698, USA. Email: tasker{at}tulane.edu
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INTRODUCTION |
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Synaptically released glutamate acts primarily on ligand-gated postsynaptic ionotropic receptors on magnocellular neurones of the hypothalamic supraoptic nucleus (SON) (Wuarin & Dudek, 1993; Jourdain et al. 1998; Stern et al. 1999). Metabotropic glutamate receptors (mGluRs) are expressed at relatively high levels in the SON (Meeker et al. 1994; van den Pol, 1994; Ohishi et al. 1995; Al-Ghoul et al. 1998), and activation of pre- and postsynaptic mGluRs in the SON with exogenous agonists elicits robust changes in transmitter release and potassium conductances, respectively (Schrader & Tasker, 1997a,b). However, little is known about the functional role of these receptors in the physiological regulation of SON neurones. Despite glutamate concentrations that reach millimolar levels in the synaptic cleft (Diamond & Jahr, 1997), pre- and postsynaptic mGluRs are usually not activated by endogenously released glutamate, due presumably to their extrasynaptic location (Baude et al. 1993; Lujan et al. 1996; although see Shigemoto et al. 1997). Under normal conditions, rapid uptake by glutamate transporters may prevent glutamate released by low-frequency activity from spreading to extrasynaptic mGluRs (Scanziani et al. 1997; Auger & Attwell, 2000). When extracellular glutamate levels are increased, however, with blockade of glutamate reuptake and/or high-frequency stimulation, the resulting extrasynaptic 'spillover' of glutamate has been reported to activate presynaptic group III mGluRs on both glutamate and 
-aminobutyric acid (GABA) terminals, leading to a reduction in release (Scanziani et al. 1997; von Gersdorff et al. 1997; Vogt & Nicoll, 1999; Dube & Marshall, 2000; Mitchell & Silver, 2000; Scanziani, 2000).
Glutamate transporters are responsible for the clearance of released glutamate and function to maintain the extracellular glutamate concentration at low, presumably subthreshold levels. Of the different subtypes of glutamate transporters, those expressed by astrocytes, the GLT-1 and GLAST transporters, are thought to play the most important role in the clearance of glutamate from around synapses, and the loss of GLT-1 and GLAST results in a several-fold increase in extracellular glutamate levels (Rothstein et al. 1996).
Under certain physiological conditions, such as with chronic dehydration and during lactation, the SON undergoes dramatic neuronal-glial morphological reorganization and synaptic restructuring (Perlmutter et al. 1985; Theodosis et al. 1986; see Hatton, 1997, for review). These changes include the retraction of astroglial processes from between magnocellular neurones and adjacent afferent terminals, and the proliferation of glutamate synapses. The retraction of astroglial processes would be expected to incur the loss of astroglial transporters and impair glutamate clearance from around synaptic terminals, and the increase in glutamate synapses would be expected to increase basal glutamate secretion, both of which should augment ambient levels of extracellular glutamate and increase activation of extrasynaptic glutamate receptors. We have found in previous studies that presynaptic group III mGluRs in the SON in slices from untreated rats are activated tonically under basal conditions (Schrader & Tasker, 1997a; Linn & Tasker, 1999). It was recently reported that the glial retraction that occurs in the SON during lactation leads to increased activation of presynaptic mGluRs by ambient glutamate (Oliet et al. 2001). In the current study, we tested whether the structural changes that occur in the SON with chronic dehydration result in an increase in the tonic activation of presynaptic mGluRs by ambient levels of glutamate using whole-cell patch-clamp recordings of miniature excitatory postsynaptic currents (mEPSCs) in SON magnocellular neurones. Some of our findings have been reported previously in abstract form (Linn & Tasker, 1999; Boudaba & Tasker, 2001).
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METHODS |
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Normal and dehydrated (2 % saline drinking water for 7-10 days) male Sprague-Dawley rats (50-120 g) were deeply anaesthetized with I.P. sodium pentobarbital (50 mg kg-1 body weight) and decapitated in a guillotine according to a protocol approved by the Tulane University Institutional Animal Care and Use Committee and in conformance with US Public Health Service guidelines. The brain was removed rapidly and placed in cold (0-2 °C) artificial cerebrospinal fluid (ACSF) bubbled with 100 % O2. The ACSF contained (mM): 140 NaCl, 3 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.4 NaH2PO4, 11 glucose, 5 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes); pH was adjusted to 7.2-7.4 with NaOH. Coronal slices (400 µm) containing the SON were sectioned and bisected along the midline, and hemi-slices were stored submerged in a holding chamber containing ACSF at room temperature (approximately 20 °C) and gently bubbled with 100 % O2 for 1.5-2 h prior to the start of experiments. One hemi-slice at a time was transferred to an interface recording chamber and continuously perfused with ACSF at 34-36 °C.
Patch electrodes (resistance 3-6 M
) were pulled from borosilicate glass (1.65 mm o.d., 1.2 mm i.d., KG-33, Garner Glass) on a Flaming-Brown horizontal puller (P-97, Sutter Instr.). The pipette solution contained (mM): 110 D-gluconic acid, 110 CsOH, 10 CsCl, 10 Hepes, 1 CaCl2, 1 MgCl2, 2 Mg-adenosine-5'-triphosphate (ATP), 0.3 sodium guanosine-5'-triphosphate (GTP), and 11 ethyleneglycol-bis[
-aminoethyl ether]-N,N,N'N'-tetraacetic acid (EGTA); pH was adjusted to 7.2-7.4 with CsOH. The osmolarity of the solution was 290-300 mosmol l-1. Caesium was included in the patch solution in place of potassium to block potassium currents.
Whole-cell recordings were performed in SON magnocellular neurosecretory neurones using the 'blind' slice patch-clamp method. Series resistance and whole-cell capacitance were monitored continually during experiments. Recorded cells were confirmed as magnocellular neurones by the presence of a large, voltage-dependent transient potassium current (Bourque, 1988) within the first ~1 min of recording, before potassium currents were blocked with the intracellular caesium perfusion.
Miniature excitatory postsynaptic currents (mEPSCs) were recorded in the presence of the sodium channel blocker, tetrodotoxin (1-1.5 µM), and the GABAA receptor antagonist, bicuculline methiodide (30 µM), at a holding potential of -60 mV with an Axopatch 1D amplifier (Axon Instruments) and were low-pass filtered at 2 kHz. All data were converted to digital video format at 22 kHz with a Neuro-Corder DR-484 digitizer unit (Neurodata Instruments Corp.) and stored on videotape for later analysis off-line. Selected data were digitized and recorded on-line on a personal computer using a Digidata 1200 interface and pCLAMP 7 software (Axon Instruments). Segments of 180 s of synaptic activity were analysed using the Mini Analysis programme (v. 3.0.1 and 4.0, Synaptosoft, Inc.). Synaptic events were selected based on crossing a threshold set empirically and were verified by eye; mEPSC amplitudes and frequencies were measured. Paired and unpaired Student's t tests were used for the intra- and intergroup statistical analyses. Probability values less than 0.05 were considered significant.
Appropriate stock solutions of mGluR agonists and antagonists and glutamate transporter blockers were prepared in distilled water and frozen at -20 °C; the stock solutions were thawed and diluted to final concentrations with ACSF just before application. The volume of the stock solution was 0.25-1 % of the total volume of ACSF, which had no effect on the osmolarity of the ACSF solution. The drugs used were L-(+)-2-amino-4-phosphonobutyric acid (L-AP4, 50-100 µM) as a group III mGluR agonist, (RS)-
-methylserine-O-phosphate (MSOP, 200 µM) as a group III mGluR antagonist, and dihydrokainate (DHK, 100-500 µM) as a glutamate transporter blocker (Tocris Cookson, Inc.). Bicuculline methiodide (30 µM) was used to block GABAA receptors and D,L-2-amino-5-phosphonovalerate (AP5, 100 µM) and 5,6-dinitroquinoxaline-2,3-dione (DNQX, 50 µM) were used to block N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic glutamate receptors (Sigma-Aldrich Co.). Tetrodotoxin (TTX, 1-1.5 µM) was used to block spike-mediated neurotransmitter release (Alomone Labs).
For immunohistochemical identification of GLT-1 transporters, rats were deeply anaesthetized with I.P. injection of sodium pentobarbital (50 mg kg-1 body weight) and perfused through the heart with 100 ml of a mixture of 4 % paraformaldehyde and 0.4 % picric acid in 0.1 M phosphate- buffered saline (PBS). The brain was removed from the cranial cavity and immersed overnight in 20 % sucrose in 0.1 M PBS. The hypothalamus was blocked with a razor and 20 µm coronal sections were cut on a cryostat. Slices containing the SON were incubated overnight at 4 °C in a guinea-pig anti-GLT-1 polyclonal antibody (1/10 000, Chemicon International Inc., CA). Sections were rinsed 3 
15 min in 0.1 M PBS and incubated for 1 h in an anti-guinea-pig IgG conjugated to fluorescein isothiocyanate (FITC, 1/200, Chemicon International Inc., CA). They were rinsed again 3 15 min in 0.1 M PBS, mounted, cover-slipped and examined with a microscope under epifluorescence illumination using a B/515 W filter combination for FITC detection.
For Western blot analyses, untreated and dehydrated rats were anaesthetized with I.P. injection of sodium pentobarbital (50 mg kg-1 body weight) and decapitated. Hypothalamic coronal slices (400 µm) were sectioned on a vibratome and stored in oxygenated ACSF at room temperature. Punches of bilateral supraoptic nuclei were collected from 2-3 slices from each rat under a dissecting microscope using a fire-polished glass capillary tube (750 µm inner diameter). Tissue samples were transferred to Eppendorf tubes containing 0.5 ml ACSF. The ACSF was removed and replaced by 50 µl lysis buffer and the tissue was sonicated for 20 s; the aliquot was removed to determine protein concentration using the colourimetric method of Lowry. Forty micrograms of total protein were loaded into each lane of a 10-lane precast 10 % tris-HCl polyacrylamide gel, electrophoresed (1 h, 125 V) and transferred to a polyvinyl difluoride membrane (BioRad, Hercules, CA, USA). Each gel was loaded with paired samples from untreated and dehydrated rats. Membranes were then incubated for 1 h at room temperature with a guinea-pig anti GLT-1 polyclonal antibody (1/1000, Chemicon International Inc., CA, USA), followed by a 30 min incubation with an alkaline phosphatase-anti-guinea-pig IgG (1/2000, Santa Cruz Inc., CA, USA). Chemiluminescence was used to detect the protein recognized by the antiserum. The blots were exposed to Kodak Biomax MR film (Health Image Kodak, NY, USA) for varying times. The GLT-1 protein was detected as a band at 42 kDa. The band densities were analysed with a model GS-700 Imaging Densitometer and Molecular Analysis V.1.4.1. Background levels were subtracted from band densities to obtain optical density measurements.
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RESULTS |
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Whole-cell patch-clamp recordings were performed in magnocellular neurosecretory cells of the supraoptic nucleus (SON) in acutely prepared hypothalamic slices (400 µm). Magnocellular neurones were voltage clamped at -60 mV in the presence of TTX (1-1.5 µM) and bicuculline methiodide (30 µM) to record mEPSCs in isolation. The mEPSCs were blocked by bath application of the ionotropic glutamate receptor antagonists AP5 (100 µM) and DNQX (50 µM) (n = 5), indicating that they were mediated by glutamate release.
Tonic activation of presynaptic mGluRs in untreated and dehydrated rats
Magnocellular neurones of the supraoptic nucleus are contacted by glutamate synaptic terminals that express group III mGluRs, the activation of which inhibits glutamate release (Schrader & Tasker, 1997a). Excitatory synaptic inputs to the magnocellular neurones are subject to a tonic inhibitory regulation by continuous activation of these presynaptic mGluRs by ambient extracellular levels of glutamate within the SON (Schrader & Tasker, 1997a; Linn & Tasker, 1999; Oliet et al. 2001). The increase in the numbers of glutamate synapses combined with the retraction of astroglial processes during chronic dehydration would be expected to result in an increase in the ambient concentration of glutamate and a corresponding increase in the tonic activation of presynaptic mGluRs. We tested this hypothesis by comparing the effects on mEPSCs of group III mGluR agonist/antagonists and a glutamate transporter blocker in slices from chronically dehydrated rats and slices from normally hydrated rats.
The effect of exogenous mGluR agonist on glutamate release should be diminished under conditions of higher basal glutamate concentration and greater presynaptic mGluR occupancy. To test this hypothesis, we compared the effect of the selective group III mGluR agonist L-AP4 in slices from dehydrated and untreated rats. Similar to a previous study in the SON (Schrader & Tasker, 1997a), bath application of L-AP4 (100 µM) resulted in a reduction of 41.7 ± 4.3 % in the frequency of mEPSCs (from 2.9 ± 0.4 Hz to 1.7 ± 0.3 Hz) in 12 of 15 cells tested (80 %; P < 0.01), without any significant effect on mEPSC amplitude (from 68.8 ± 33.8 pA to 57.6 ± 28.6 pA; P = 0.09), in SON neurones from untreated rats. The range of decrease in mEPSC frequency caused by L-AP4 in cells from untreated animals was 14 % to 65 %. The effect of L-AP4 was attenuated in slices from dehydrated rats, causing a 27.8 ± 4.2 % reduction in mEPSC frequency (from 6.4 ± 1.5 Hz to 4.6 ± 1.0 Hz) in 9 of 13 cells tested (69.2 %; P < 0.01), again with no effect on mEPSC amplitude (from 86.8 ± 22 pA to 84.6 ± 30.3 pA; P = 0.5) (Fig. 1). The range of decrease in mEPSC frequency caused by L-AP4 in cells from dehydrated animals was 11 % to 48 %. The significantly reduced effect of the group III mGluR agonist on glutamate release in the SON in chronically dehydrated rats compared with untreated rats (P < 0.05) suggested an increase in the basal occupancy of presynaptic mGluRs by ambient glutamate with dehydration.
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Figure 1. Effect of an exogenous group III mGluR agonist on glutamate release in untreated and dehydrated rats A, application of L-AP4 (100 µM) caused a decrease in the frequency of mEPSCs in representative SON neurones recorded in slices from untreated (1) and dehydrated rats (2). B, average percentage change in mean mEPSC frequency caused by L-AP4 (100 µM) in SON neurones from untreated and dehydrated rats. The average change in mEPSC frequency caused by L-AP4 was attenuated in SON neurones from dehydrated rats (*P < 0.05, Student's t test). | ||
Bath application of the group III mGluR antagonist MSOP (200 µM) in slices from untreated rats elicited a non-significant increase in mEPSC frequency of 31.4 ± 3.2 % (from 2.8 ± 1.2 Hz to 4.1 ± 1.9 Hz) in 6 of 9 cells tested (66.6 %, P = 0.07). It had no effect on mEPSC amplitude (from 107.3 ± 40.4 pA to 108.6 ± 42.5 pA; P = 0.7). The range of decrease in mEPSC frequency caused by MSOP in cells from untreated animals was 11 % to 37 %. In slices from dehydrated rats, MSOP (200 µM) caused a 42.8 ± 5.4 % increase in mEPSC frequency (from 3.0 ± 0.8 Hz to 4.8 ± 1.0 Hz) in 11 of 15 neurones tested (73.3 %, P < 0.01), with no effect on mEPSC amplitude (from 107.0 ± 32.0 pA to 114.2 ± 34 pA; P = 0.4) (Fig. 2). The range of decrease in mEPSC frequency caused by MSOP in cells from dehydrated rats was 22 % to 73 %. The facilitatory effect of the mGluR antagonist on glutamate release was significantly higher in slices from dehydrated rats than in slices from untreated rats (P < 0.05). These data indicate that presynaptic group III mGluRs are tonically activated by ambient glutamate in the SON and that this activation is enhanced in slices from dehydrated rats, again suggesting a higher basal occupancy of mGluRs by ambient glutamate.
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Figure 2. Effect of blockade of presynaptic group III mGluRs on glutamate release onto SON neurones in slices from untreated and dehydrated rats A, application of MSOP (200 µM) caused an increase in the frequency of mEPSCs in representative SON neurones from untreated (1) and dehydrated (2) rats, indicating a basal activation of presynaptic mGluRs by ambient glutamate. B, average percentage change in the mean frequency of mEPSCs induced by MSOP in SON neurones from untreated and dehydrated rats. The average increase in mean mEPSC frequency was greater in SON neurones from dehydrated rats (*P < 0.05, Student's unpaired t test). | ||
Reducing glutamate uptake by blocking astrocytic glutamate transport increases the tonic activation of presynaptic mGluRs in the SON (Linn & Tasker, 1999; Oliet et al. 2001), due presumably to an increase in ambient glutamate levels. We postulated that this effect would be reduced in the SON of dehydrated animals due to a higher ambient glutamate concentration resulting from basal release from an increased number of glutamate synapses and from reduced glutamate uptake caused by the retraction of astrocytic processes. In slices from untreated rats, dihydrokainate (DHK, 500 µM), a selective GLT-1 transporter blocker (Arriza et al. 1994; Rothstein et al. 1994), reduced the frequency of mEPSCs by 48.8 ± 5.2 % (from 5.0 ± 2.4 Hz to 2.7 ± 1.5 Hz) in 8 of 11 neurones tested (72.7 %, P < 0.05), without affecting the amplitude of mEPSCs (from 108.6 ± 39.0 pA to 117.7 ± 38 pA; P = 0.2). The range of decrease in mEPSC frequency caused by DHK in cells from untreated animals was 26 % to 73 %. A lower concentration of DHK (100 µM) caused a similar reduction (45.0 ± 7.8 %) in mEPSC frequency (from 7.5 ± 3.1 Hz to 3.7 ± 1.0 Hz) in 3 of 3 cells tested. The percentage change of the frequency of mEPSCs at this frequency was not significantly different from that induced by 500 µM DHK (P = 0.7), indicating that the effect was not due to agonist actions at kainate/AMPA receptors (Kidd & Isaac, 2000). The effect of DHK on mEPSCs was blocked completely by prior blockade of the group III mGluRs with MSOP (200 µM) or by saturation of the group III mGluRs with L-AP4 (100 µM) (data not shown), which indicated that it was mediated by activation of presynaptic group III mGluRs. The effect of DHK on mEPSCs was weaker in slices from dehydrated rats (Fig. 3), resulting in a reduction in the frequency of mEPSCs of 35.4 ± 4.7 % (from 3.9 ± 1.0 Hz to 2.8 ± 0.9 Hz) in 12 of 13 neurones tested (92.3 %, P < 0.01). The range of decrease in mEPSC frequency caused by DHK in cells from dehydrated animals was 6 % to 61 %. Again, DHK had no effect on mEPSC amplitude (from 35.1 ± 14.4 pA to 29.5 ± 9.4 pA; P = 0.2). The reduced effect of glutamate transporter blockade on glutamate release in dehydrated rats compared with untreated rats (P < 0.05) suggested a diminished influence of glutamate uptake on ambient glutamate concentration and presynaptic mGluR activation in chronically dehydrated rats.
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Figure 3. Effect of blockade of glutamate reuptake on glutamate release in slices from untreated and dehydrated rats A, application of the GLT-1 transporter blocker DHK (500 µM) caused a decrease in the frequency of mEPSCs in representative SON neurones from an untreated (1) and a dehydrated rat (2), suggesting an enhanced tonic activation of presynaptic mGluRs by increased ambient glutamate levels during dehydration. B, average percentage decrease in the mean frequency of mEPSCs induced by DHK in SON neurones from untreated and dehydrated rats. The average decrease in mean mEPSC frequency caused by blocking glutamate reuptake was attenuated in SON neurones from dehydrated rats (*P < 0.05, Student's t test). | ||
Reduced expression of GLT-1
Immunohistochemical and Western blot techniques were used to localize and confirm the presence of the glial GLT-1 transporter in the SON of untreated rats, and to determine whether GLT-1 protein expression changes with chronic dehydration. In untreated rats, robust GLT-1 immunoreactivity was found distributed diffusely throughout the SON, surrounding the somata of the magnocellular neurones (Fig. 4). Although not analysed further, this pattern of labelling is consistent with a non-neuronal expression of the GLT-1 transporter (Arriza et al. 1994; Rothstein et al. 1994) and with a prominent role of the GLT-1 transporter in the clearance of synaptically released glutamate in the SON.
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Figure 4. GLT-1 transporter protein expression in SON and immunohistochemical localization of the GLT-1 subtype of glutamate transporter in the SON A, low-power photomicrograph showing GLT-1 immunoreactivity distributed throughout the SON. B, at higher magnification, labelling is seen distributed in a peri-cellular fashion (arrows). OC, optic chiasm. C, immunoblot analysis of GLT-1 protein expression in SON tissue punches from four untreated rats (U1-U4) and four dehydrated rats (D1-D4). D, relative expression of GLT-1 in SON punches from untreated and dehydrated rats from immunoblots shown in C. The mean intensity of the GLT-1 expression in SON punches from dehydrated rats was normalized to the GLT-1 expression in SON punches from untreated rats (**P < 0.01, Student's unpaired t test). | ||
Comparison of GLT-1 protein expression levels with Western blot analysis of SON punches revealed a significant down-regulation of the GLT-1 transporter in the SON with dehydration (Fig. 4).
The trunk blood osmolality of dehydrated rats at the time of sacrifice was 391.1 ± 8.26 mosmol kg-1, while that of normally hydrated rats was 300.0 ± 1.5 mosmol kg-1 (n = 15, P < 0.01).
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DISCUSSION |
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Studies of activation of mGluRs with subtype-selective agonists have consistently found a role of the group III mGluRs in the regulation of glutamate and GABA release from presynaptic terminals in different areas of the brain, including the SON (see Conn & Pin, 1997, for review; Schrader & Tasker, 1997a). However, activation of presynaptic mGluRs by synaptically released glutamate is usually seen only under conditions of glutamate 'spillover' caused by high-frequency stimulation and/or blockade of glutamate reuptake (Scanziani et al. 1997; von Gersdorff et al. 1997; Vogt & Nicoll, 1999). This high level of synaptically released glutamate necessary to activate presynaptic mGluRs is inconsistent with the immunohistochemical localization of mGluR7a within the presynaptic active zones of putative glutamatergic and GABAergic synapses in the hippocampus (Shigemoto et al. 1997). We reported in a previous study an enhancement of electrically evoked EPSCs in SON magnocellular neurones induced by blocking presynaptic group III mGluRs (Schrader & Tasker, 1997a), which suggested that these receptors might be tonically activated by endogenous ligand. We confirm here that the presynaptic group III mGluRs on glutamate terminals are indeed activated tonically by ambient levels of glutamate in the SON in our slice preparation, and that this basal activation of presynaptic mGluRs is augmented during chronic dehydration.
We found that blockade of glutamate transporter activity by a selective GLT-1-type transporter (DHK) had an effect on glutamate release that was similar to that caused by the activation of group III mGluRs with an exogenous agonist, L-AP4. Interestingly, GLT-1 transporter blockade decreased glutamate release to a degree comparable to that caused by the activation of the presynaptic mGluRs with a near-saturating concentration of L-AP4 (i.e. by 42 % with L-AP4 compared with 49 % with DHK). This suggests that the GLT-1 subtype of transporter, which is expressed primarily by glial cells (Rothstein et al. 1994), plays a prominent role in determining the ambient glutamate concentration in the SON, but also that blocking glutamate reuptake can result in elevating ambient glutamate to a level high enough to saturate presynaptic group III mGluRs. The important role of the GLT-1 transporter in regulating glutamate clearance is consistent with findings from in vivo antisense experiments in which it was primarily the GLT-1 transporter, and not the GLAST or EAAC1 subtypes of transporter, that was found to control extracellular glutamate levels in the striatum (Rothstein et al. 1996). A role for the GLT-1 transporter in buffering the extracellular glutamate levels bathing magnocellular neurones is further supported by our finding of a dense, peri-cellular distribution of GLT-1 immunoreactivity in the SON.
Ambient extracellular glutamate concentration has been reported in the submicromolar concentration range (Rothstein et al. 1996; Zerangue & Kavanaugh, 1996), and extrasynaptic glutamate concentration increases in the hippocampus to 160-190 µM with low-frequency electrical stimulation (Dzubay & Jahr, 1999). If similar extracellular glutamate levels were present in the hypothalamus, our findings would suggest that the presynaptic mGluRs in the SON are activated by submicromolar concentrations of ambient glutamate, which is consistent with a presynaptic mGluR localization in close proximity to synaptic release sites (Shigemoto et al. 1997). However, the lack of tonic activation of presynaptic mGluRs in the hippocampal CA1 subfield (Bergles & Jahr, 1997) and in the locus coeruleus (Dube & Marshall, 2000) suggests that the extracellular ambient glutamate levels in the SON may be higher than in these other structures, at least in the vicinity of the presynaptic mGluRs, or that the presynaptic mGluRs in the SON have a higher affinity for glutamate. Interestingly, hypothalamic cultures containing both neurones and glia have been found to have a greater glutamate buffering capacity than cortical cultures (Pak & Curras-Collazo, 1999), suggesting that a higher ambient glutamate concentration may not be responsible for the higher level of activation of presynaptic mGluRs in the SON.
The tonic activation of presynaptic mGluRs by ambient glutamate reduces the probability of release from individual glutamatergic synapses, thereby exerting a negative feedback effect on basal glutamate release. This feedback regulation of glutamate release should effectively reduce the baseline synaptic noise in magnocellular neurones caused by quantal glutamate release. Since this low level of negative regulation would not be expected to affect release mediated by action potential invasion of presynaptic glutamate terminals, its role might be to increase the signal-to-noise ratio in the postsynaptic magnocellular neurones, as suggested by Oliet and colleagues (2001).
Increased mGluR activation with chronic dehydration
The ambient glutamate concentration, and thus the degree of tonic activation of presynaptic mGluRs, is determined by the level of glutamate release and by the efficiency of glutamate clearance by glutamate transporters. The structural reorganization of the SON caused by chronic dehydration includes glutamate synapse proliferation and retraction of astroglial processes from around magnocellular neurones (Theodosis & Poulain, 1993; Hatton, 1997), which would be expected to both increase quantal glutamate release and reduce glutamate clearance, respectively. We have preliminary evidence that quantal glutamate release in the SON increases with chronic dehydration (Di & Tasker, 1999), consistent with an increase in synapse numbers and an increase in ambient levels of glutamate. Our present study shows that, in slices from dehydrated rats, agonists of presynaptic mGluRs in the SON have a lesser effect, presynaptic mGluR antagonists are more effective, and the effect of blocking glutamate uptake with a glutamate transporter blocker is diminished compared with slices from untreated rats. Together these findings suggest that dehydration causes an increase in the basal activation of presynaptic mGluRs by increasing ambient glutamate levels. This is consistent with observations in slices from lactating rats (Oliet et al. 2001), which show anatomical changes that are similar to those seen with dehydration (Theodosis & Poulain, 1993; Hatton, 1997). In addition to the anatomical rearrangement of the SON during chronic dehydration, our findings showed a reduction of GLT-1 transporter protein expression by glial cells. The down-regulation of the GLT-1 transporter with dehydration should lead to a reduction in glutamate uptake and contribute to the accumulation of glutamate in the extracellular space.
If tonic activation of presynaptic mGluRs by ambient glutamate serves a negative feedback role to reduce basal glutamate release and decrease synaptic noise in magnocellular neurones, then our findings suggest that this function is enhanced following chronic dehydration. The result of the increased negative feedback would be to decrease the release probability at individual glutamate synapses. This decrease in the probability of release might serve a compensatory role to prevent an increase in quantal release, and resulting synaptic noise, by the increased numbers of glutamate release sites that occur with chronic dehydration.
Significance for magnocellular excitability
We demonstrated previously that postsynaptic group I mGluRs and presynaptic group III mGluRs in the SON are activated by application of exogenous ligands and cause, respectively, the depolarization of magnocellular neurones by inhibiting potassium currents (Schrader & Tasker, 1997b) and the suppression of glutamate and GABA release in the SON (Schrader & Tasker, 1997a). Our present findings show that the presynaptic group III mGluRs on glutamate terminals are activated tonically by ambient levels of endogenous glutamate, and that this tonic activation is augmented following chronic dehydration. A similar increase in tonic activation of presynaptic mGluRs by ambient glutamate has been reported in slices from lactating rats (Oliet et al. 2001), in which comparable structural modifications of the SON occur (Theodosis & Poulain, 1993; Hatton, 1997). As mentioned above, this auto-regulation by glutamate should serve to reduce the synaptic noise that would otherwise occur with increased numbers of glutamate synapses. Additionally, the increased level of activation of presynaptic mGluRs and resulting decrease in glutamate release probability should result in lower levels of glutamate in the synaptic cleft, and therefore in less glutamate available to bind to postsynaptic ionotropic receptors. A lower level of tonic activation of postsynaptic ionotropic receptor might be expected to decrease ionotropic receptor desensitization, and lead, therefore, to an enhancement of postsynaptic responsiveness to synaptic glutamate release. Although mGluR activation appears not to influence the oxytocin and vasopressin secretory response to acute osmotic stimulation in vitro (Morsette et al. 2001; Meeker, 2002), our findings suggest that these receptors may have an increased influence on hormone release in chronically dehydrated animals.
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Acknowledgements
We dedicate this paper to the memory of Kriszta Szabó, whose passing has touched us profoundly. This research was supported by grants from the National Institutes of Health (NS34926 and NS042081) and American Heart Association (96010150). We are grateful to Dr Gabor Halmos for the densitometry readings, and to Drs Andrei Belousov and Bret Smith for having read and critiqued an early draft of the manuscript.
Author's present address
D. M. Linn: Pharmacia Corporation, CNS Discovery, Kalamazoo, MI 49007, USA.
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