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Division of Neurobiology, Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana 70118-5698
Address all correspondence and requests for reprints to: Jeffrey G. Tasker, Ph.D., Department of Cell and Molecular Biology, 2000 Percival Stern Hall, Tulane University, New Orleans, Louisiana 70118-5698. E-mail: tasker{at}mailhost.tcs.tulane.edu.
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Abstract |
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-aminobutyric acid (GABA) release. Hypothalamic magnocellular neuroendocrine cells undergo dramatic changes in synaptic organization under conditions of increased hormone release, including increased numbers of glutamatergic, GABAergic and noradrenergic synapses. We studied the functional plasticity of magnocellular neurons of the rat supraoptic nucleus induced by chronic dehydration using whole-cell recordings in hypothalamic slices. Dehydrated rats showed increases in glutamate and GABA release onto magnocellular neurons, as evidenced by an increase in the frequency of spontaneous excitatory (29%) and inhibitory (33%) postsynaptic currents. The change in glutamate release was likely due to increased numbers of release sites because paired-pulse facilitation analysis did not reveal a change in the probability of transmitter release. In untreated rats, norepinephrine facilitates glutamate release and attenuates GABA release onto magnocellular neurons. Dehydration resulted in a marked enhancement of norepinephrine’s actions, doubling both the norepinephrine-induced increase in glutamate release and decrease in GABA release. The norepinephrine dose-response curve was shifted to the left with dehydration, revealing an increase in norepinephrine sensitivity. Thus, dehydration leads to an increase in glutamate and GABA release onto supraoptic magnocellular neurons as well as a marked enhancement of the facilitatory effect of norepinephrine on glutamate release and inhibitory effect on GABA release. This synaptic plasticity would be expected to increase the excitability of the magnocellular neurons and support the enhanced bursting capacity and facilitated hormone secretion observed in vivo with chronic dehydration. |
Introduction |
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-aminobutyric acid (GABA)ergic (6, 7), and noradrenergic synapses (5) impinging on the magnocellular neurons are all involved in the morphological changes that occur during chronic physiological stimuli (3). Recent in vitro electrophysiological studies have demonstrated corresponding functional changes in the glutamatergic and GABAergic synaptic inputs to SON magnocellular neurons during lactation (8, 9, 10).
Previous studies demonstrated that norepinephrine has an excitatory effect on the magnocellular neuroendocrine cells of the SON and PVN under normal conditions by acting at both postsynaptic and presynaptic receptors. Postsynaptically, norepinephrine causes a depolarization and an increase in firing in both magnocellular neurons (11, 12) and upstream local glutamatergic interneurons in the PVN (13) via 
1-receptor activation. Norepinephrine also causes an increase in glutamate release and a decrease in GABA release in the SON and PVN by acting at 1 and 2 receptors on presynaptic terminals (14, 15, 16). The present experiments were designed to study the physiological consequences of the structural synaptic changes seen in the glutamatergic, GABAergic and noradrenergic innervation of rat SON magnocellular neurons with chronic dehydration. We found that, after chronic dehydration, the spontaneous release of glutamate and GABA was increased and that both the presynaptic and postsynaptic effects of norepinephrine were markedly enhanced.
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Materials and Methods |
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Slice preparation
Rats were deeply anesthetized with sodium pentobarbitol (50 mg/kg body weight) and decapitated. The brain was quickly removed from the cranial cavity after cutting the optic nerves and was immersed in a cooled (1–2 C), oxygenated (100% O2) artificial cerebral spinal fluid mM (aCSF). The composition of the aCSF was (in millimoles): 140 NaCl, 3 KCl, 1.3 MgSO4, 1.4 NaH2PO4, 2.4 CaCl2, 11 glucose, and 5 HEPES (pH was adjusted to 7.2–7.3 with NaOH). The hypothalamus was blocked, and the caudal end of the block was glued to the chuck of a vibrating microtome (World Precision Instruments, Sarasota, FL). Two coronal hypothalamic slices (350–400 µm) containing the SON were sectioned, bisected along the midline (i.e. along the third ventricle), and submerged in a holding chamber in oxygenated aCSF at room temperature in which they were allowed to equilibrate for at least 1.5 h before being transferred to a recording chamber.
Electrophysiological methods
Patch pipettes were pulled from borosilicate glass (1.65 mm outer diameter, 1.2 mm inner diameter; KG33; Garner Glass, Claremont, CA) with a Flaming/Brown P-97 micropipette puller (Sutter Instruments, Novato, CA) to a resistance of 3–4 M
. They were coated at the tip with silicone polymer (Sylgard) to reduce electrode capacitance and filled mM with a solution containing (in millimoles): 10 CsCl, 110 CsOH, 110 D-gluconic acid, 10 HEPES, 1 MgCl2, 1 CaCl2, 11 EGTA, 2 Mg-ATP, and 0.3 Na-GTP (pH was adjusted to 7.2–7.3 with CsOH). The osmolarity of the solution was adjusted to 300 mOsm with D-sorbitol.
Slices were transferred to a submersion recording chamber and allowed to equilibrate for at least 15 min before recording. Magnocellular neurons in the SON were visualized directly via a cooled CCD camera using infrared illumination and differential interference contrast optics and were identified based on their shape, size, and location within the slices. Only one cell was recorded in each hemislice. Whole-cell recordings were conducted at 22–24 C; series resistance was compensated by 80%, and changes were monitored and compensated throughout the recordings. The average series resistance at the beginning of recordings was 5.6 ± 0.4 M in the untreated group and 4.4 ± 0.3 M in the dehydrated group. Recordings with an unstable series resistance or neuronal input resistance were not retained in the study. All recordings were performed in voltage clamp mode using an Axopatch 1-D amplifier (Axon Instruments, Foster City, CA) and were monitored continuously on a digital storage oscilloscope (Hitachi, Tokyo, Japan). Data were low-pass filtered at 2 kHz, converted to digital video format at 22 kHz (Neuro Corder, Cygnus Technologies, Delaware Water Gap, PA), and stored on videotape for off-line analysis. Selected data were digitized at 4 kHz and recorded on line using the Digidata 1200 interface and pCLAMP 7.0 software (Axon Instruments). Periods of 180 sec of synaptic activity were recorded at a holding potential of 0 mV for inhibitory postsynaptic currents (IPSCs) and –60 mV for excitatory postsynaptic currents (EPSCs). The traces obtained were analyzed using the Minianalysis 4.0 program (Synaptosoft Inc., Decatur, GA).
To study paired-pulse facilitation, a bipolar tungsten stimulation electrode was placed dorsal to the SON with 0.5 mm separation between the two poles. Two excitatory synaptic responses (S1 and S2) were evoked every 10 sec by a pair of stimuli (0.5 msec, 0.2–0.4 mA) delivered at a 60-msec interval. Paired-pulse facilitation was expressed as the ratio of the amplitude of the second synaptic response to the amplitude of the first synaptic response (S2/S1).
Statistical analyses were performed using the Student’s paired t test for within-group comparisons and the Student’s unpaired t test for between-group comparisons. Significance was set at P < 0.05. Data are expressed as means ± SE.
Intracellular labeling and immunohistochemistry
Some cells were injected with biocytin (0.1–0.3% in patch electrodes) as an intracellular marker during recordings (17). After experiments, slices were fixed overnight in 4% paraformaldehade in 0.1 M PBS. Intracellular biocytin was labeled by incubation of free-floating sections in streptavidin-conjugated 7-amino-4-methyl-coumarin-3-acetic acid (AMCA, Molecular Probes, Eugene, OR), diluted (1:500) in 0.1 M PBS containing 1% Triton X-100. Sections were screened for biocytin-filled, AMCA-labeled cells with an epifluorescence microscope, and sections containing labeled cells were subjected to an immunofluorescence labeling procedure. Briefly, slices were incubated in a mixture of a rabbit polyclonal antibody to oxytocin-associated neurophysin (VA10) diluted 1:2000,and a mouse monoclonal antibody to VP-associated neurophysin (PS-41) diluted 1:4000 in 0.1 M PBS + 1% normal sheep serum and 0.2% sodium azide (antibodies kindly provided by Dr. H. Gainer, National Institutes of Health, Bethesda, MD). After PBS rinses, the sections were incubated in goat antirabbit secondary IgG conjugated with fluorescein isothiocyanate (FITC) and goat antimouse IgG secondary conjugated with rhodamine (Vector Laboratories, Burlingame, CA) for 1 h and rinsed in PBS again. Slices were mounted and coverslipped with Vectashield antifading mounting medium (Vector Laboratories) and examined for biocytin labeling and OT or VP immunofluorescence.
Drug application
Norepinephrine, tetrodotoxin, glutamate receptor antagonists, and the GABA receptor antagonist were dissolved in aCSF and applied in the perfusion bath. Norepinephrine (Sigma, St. Louis, MO) was applied at concentrations ranging from 0.1 µM to 1 mM. Glutamate receptor antagonists included the N-methyl-D-aspartate (NMDA) receptor antagonist D, L-2-amino-5-phosphonovalerate (AP5, 50–100 µM), and the non-NMDA receptor antagonist 5,6-dinitroquinoxaline-2,3-dione (DNQX, 30–50 µM) (Tocris Cookson, Ballwin, MO). The GABAA receptor antagonist used was bicuculline methiodide (30 µM; Sigma). Tetrodotoxin (TTX, 1–3 µM; Sigma) was used to block voltage-gated Na+ channels and action potential-mediated transmitter release.
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Results |
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A total of 140 putative magnocellular neurons were recorded in hypothalamic slices from 84 rats, including 73 neurons from the dehydrated group and 67 neurons from the untreated controls. The mean input resistance was similar for the two groups (untreated: 1.7 ± 0.1 G, dehydrated: 1.7 ± 0.1 G; P = 0.37). Based on a qualitative visual assessment, the somata of the magnocellular neurons from the dehydrated rats were enlarged, compared with those from the untreated rats. Consistent with a larger somatic size, magnocellular neurons from dehydrated rats showed a 20% increase in whole-cell capacitance (untreated: 7.8 ± 0.5 pF, dehydrated: 9.4 ± 0.6 pF; P < 0.05). The holding current required to clamp the cells at –60 mV was similar in the two groups (untreated: –41.3 ± 2.2 pA; dehydrated: –43.6 ± 2.8 pA; P = 0.26). Spontaneous EPSCs were blocked completely by the glutamate receptor antagonists AP-5 (100 µM) and DNQX (50 µM) (n = 4) but were not affected by the GABAA receptor antagonist bicuculline methiodide (30 µM) (n = 16), indicating that they were mediated by synaptically released glutamate. The spontaneous IPSCs were blocked completely by the GABAA receptor antagonist bicuculline methiodide (30 µM) (n = 3) but were not affected by the glutamate receptor antagonists AP-5 (50 µM) and DNQX (30 µM) (n = 3), indicating that they were elicited by synaptic GABA release.
Effect of dehydration on postsynaptic norepinephrine actions
Bath application of norepinephrine at concentrations 10 µM or more generally elicited an inward membrane current, which was larger in neurons from dehydrated rats. At a holding potential of –60 mV, norepinephrine (10 µM) caused an inward current of 25.9 ± 3.8 pA in neurons from the untreated group (n = 38), which increased to 82.4 ± 9.6 pA in neurons from the dehydrated group (n = 23), representing a 218% increase (P < 0.01). The norepinephrine-evoked inward membrane current was not blocked by AP-5 (100 µM) and DNQX (50 µM) in cells from the untreated group (from 29.3 ± 10.7 to 26.4 ± 12.3 pA, n = 7, P = 0.45) or cells from the dehydrated group (89.2 ± 14.4 to 83.7 ± 17.9 pA, n = 6, P = 0.47), indicating that it was not mediated by ionotropic glutamate receptor activation. The greater change in the norepinephrine-induced, glutamate-independent inward current in the dehydrated group suggested a more robust postsynaptic effect of norepinephrine in SON magnocellular neurons after chronic dehydration. There was no significant change in input resistance elicited by norepinephrine application in either of the two groups (untreated: 1.5 ± 0.2 to 1.3 ± 0.1 G, n = 14, P = 0.3; dehydrated: 1.3 ± 0.2 to 1.2 ± 0.2 G, n = 9, P = 0.24).
Effect of dehydration on glutamate release
Spontaneous EPSCs were analyzed in a total of 91 SON magnocellular neurons, 44 neurons in slices from dehydrated rats, and 47 neurons in slices from control rats. Dehydrated rats showed an increase in the frequency, amplitude, and decay kinetics of EPSCs, compared with untreated rats (Fig. 1
). In recordings in slices from control rats, the mean EPSC frequency was 2.1 ± 0.2 Hz (range 0.3–7.1 Hz); the mean EPSC amplitude was 25.1 ± 1.2 pA (range 11.6–44.1 pA); and the mean decay time of averaged EPSCs, defined as the time from peak to the point at which the EPSC had decayed by 63%, was 3.6 ± 0.2 ms (range 2.0–4.9 ms). In the neurons from dehydrated rats, the mean EPSC frequency was increased by 29%, to 2.7 ± 0.2 Hz (range 0.9–5.4 Hz; P < 0.05), the mean EPSC amplitude was increased by 17%, to 29.4 ± 1.2 pA (range 10.2–46.9 pA; P < 0.05), and the mean EPSC decay time was decreased by 32%, to 2.4 ± 0.1 ms (range 1.5–4.1 ms; P < 0.01).
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). The synaptic responses from 50 to 100 consecutive paired-pulse trials, including synaptic failures, were averaged. The mean amplitudes of the first evoked EPSCs (S1) were 37.0 ± 9.4 and 45.7 ± 12.6 pA in five neurons from four untreated rats and in seven neurons from five dehydrated rats, respectively, which were not significantly different from one another (P = 0.31, Student’s unpaired t test). The mean amplitudes of the second evoked EPSCs (S2) were 44.4 ± 10.1 and 53.0 ± 15.5 pA in neurons from the untreated and the dehydrated rats, respectively. The average paired-pulse ratios were 1.26 ± 0.09 in neurons from the untreated group and 1.15 ± 0.03 in neurons from the dehydrated group, which were not significantly different (P = 0.11, Student’s unpaired t test). This suggests that the observed change in glutamate release was not caused by an increase in the probability of glutamate release but was due, rather, to an increase in the number of glutamate synapses in the dehydration model.
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). In neurons from the control group (n = 20), the mean IPSC frequency was 1.7 ± 0.3 Hz (range 0.4–3.9 Hz); the mean amplitude of IPSCs was 55.8 ± 5.5 pA (range 18.1–97.4 pA); and the mean IPSC decay time, defined as the time from peak to the point at which the IPSC had decayed by 63%, was 4.9 ± 0.1 ms (range 2.9–5.2 ms). There was a 33% increase in the frequency of IPSCs in the neurons from the dehydrated group (n = 29), to 2.2 ± 0.2 Hz (range 1.1–4.5 Hz; P < 0.05). There was no significant change in the mean IPSC amplitude (62.1 ± 3.5 pA; range 24.1–99.3 pA; P = 0.16) or decay time (5.1 ± 0.1 ms; range 3.5–5.2 ms; P = 0.11). None of the parameters of either the spontaneous EPSCs or IPSCs were affected by blocking spike-mediated transmitter release with bath application of TTX (1–3 µM) in neurons from either the untreated group or the dehydrated group (data not shown, n = 4 from each group for all parameters).
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-receptor-mediated increase in glutamate release (13, 14). Here norepinephrine caused a markedly enhanced increase in EPSC frequency in SON neurons recorded in slices from dehydrated rats (Fig. 4). The mean EPSC frequency increased in 10 µM norepinephrine by 428% in neurons from dehydrated rats (from 2.4 ± 0.5 to 12.9 ± 4.1 Hz, n = 8, P < 0.05), compared with a 223% increase in EPSC frequency in neurons from untreated rats (from 2.0 ± 0.4 to 6.6 ± 1.7 Hz, n = 8, P < 0.05), which represents a significant change in the norepinephrine response between the two groups (P < 0.01). In contrast, the mean amplitude and decay time of EPSCs were not affected significantly by norepinephrine in either the dehydrated or untreated animals. To determine whether the change in the noradrenergic modulation of glutamate release in dehydrated rats was due to changes in noradrenergic sensitivity at presynaptic glutamatergic terminals or at presynaptic local glutamate circuit neurons (13, 14), we tested the norepinephrine response for its dependence on spike generation. A 10-min application of TTX (1–3 µM) and norepinephrine (10 µM) after a 10-min exposure to norepinephrine alone (10 µM) had no effect on the norepinephrine-evoked increase in EPSC frequency in magnocellular neurons from either the untreated (5.0 ± 1.1 Hz in norepinephrine vs. 4.8 ± 1.1 Hz in norepinephrine and TTX, n = 7, P = 0.28) or dehydrated rats (11.8 ± 2.4 Hz in norepinephrine vs. 11.5 ± 2.4 Hz in norepinephrine and TTX, n = 8, P = 0.33).
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). The threshold concentration of norepinephrine decreased from 10 µM in the untreated group to 1 µM in the dehydrated group. The differences in the response to norepinephrine between the untreated and dehydrated groups reached statistical significance at 1 (P < 0.05), 10 (P < 0.01), and 100 µM (P < 0.05).
Effect of dehydration on the norepinephrine-induced decrease in GABA release
Previous studies showed that norepinephrine causes a decrease in the frequency of spontaneous IPSCs in SON and PVN magnocellular neurons by reducing GABA release from presynaptic terminals (15, 16). Here we also tested the norepinephrine effect on spontaneous IPSCs in SON magnocellular neurons in slices from both dehydrated and untreated rats. Norepinephrine (100 µM) caused a 39% decrease in the frequency of IPSCs in SON neurons from untreated animals (from 1.7 ± 0.3 to 1.0 ± 0.2 Hz, n = 23, P < 0.05). The same concentration of norepinephrine elicited a greater response in SON neurons from dehydrated rats, causing a 57% decrease in the mean frequency of IPSCs (from 2.0 ± 0.2 to 0.8 ± 0.1 Hz, n = 28, P < 0.05) (Fig. 5), which represents a significant change in the norepinephrine response between the untreated and dehydrated groups (P < 0.01). In contrast, no effect of norepinephrine on the mean amplitude or mean decay time of IPSCs was observed in neurons recorded in slices from either untreated or dehydrated rats. Blocking spike-mediated transmitter release by adding TTX (1–3 µM, 10 min) to the norepinephrine after 10 min of norepinephrine alone had no effect on the norepinephrine-induced decrease in GABA release in magnocellular neurons from either untreated (0.74 ± 0.08 Hz in norepinephrine vs. 0.70 ± 0.09 Hz in norepinephrine and TTX, n = 8, P = 0.55) or dehydrated rats (0.92 ± 0.19 Hz in norepinephrine vs. 0.89 ± 0.17 Hz in norepinephrine and TTX, n = 10, P = 0.22).
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). In neurons from untreated rats, a decrease in the frequency of IPSCs to 74 ± 6% of control was observed with a threshold norepinephrine concentration of 10 µM (n = 3), whereas in neurons from the dehydrated rats, a threshold norepinephrine concentration of 1 µM reduced the mean frequency of IPSCs to 80 ± 7% of control (n = 5). The differences in the response to norepinephrine between the untreated and dehydrated groups reached statistical significance at 1 (P < 0.05), 10 (P < 0.05), and 100 µM (P < 0.01).
Immunohistochemical identification of magnocellular neurons
After recordings of the effect of norepinephrine on EPSCs, 11 magnocellular neurons were labeled with biocytin and were immunopositive for one and immunonegative for the other of the two antibodies directed against OT- and VP-associated neurophysins (i.e. 12% of a total of 91 neurons filled with biocytin and processed immunohistochemically). Of seven neurons from the untreated rats, three were immunopositive for OT, and four were immunopositive for VP. Of four cells from the dehydrated group, three were immunopositive for OT, and one was immunopositive for VP. As shown in Table 1, the identified OT and VP neurons showed similar responses in inward current and EPSC frequency to NE in untreated and dehydrated groups. These results suggest that the observed changes in synaptic inputs to magnocellular neurons with dehydration are not specific to one or the other of the two SON cell populations.
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Discussion |
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Presynaptic changes
We recorded significant increases in the average frequencies of spontaneous EPSCs (29%) and IPSCs (33%) in SON magnocellular neurons after 7–10 d of chronic dehydration. The EPSCs and IPSCs were mediated by glutamate and GABA release, respectively, and the increase in synaptic activity in the SON, therefore, represents an increase in the release of glutamate and GABA with dehydration. The increase in glutamate release appears to be caused by the increased numbers of glutamate synapses reported in morphological studies (18, 24) and not by an increase in the probability of glutamate release from a constant number of synapses because there was no significant change in paired-pulse facilitation, a measure of release probability (25), in cells from dehydrated rats. Indeed, based on recent findings from our laboratory, we would expect to find a decrease in the release probability due to the increase in autoinhibitory presynaptic metabotropic glutamate receptor activation caused by increased ambient glutamate levels in the SON of dehydrated rats (26). Therefore, the data presented here suggest that the increases in glutamatergic and GABAergic synaptic inputs to magnocellular neurons of the SON are the physiological correlate to the increased numbers of glutamate and GABA synapses seen on these cells after dehydration. Increases in glutamate and GABA synapses in the SON have also been reported during lactation (for review, see Refs. 2 and 3), and recent electrophysiological studies have described similar, albeit more robust, increases in glutamatergic EPSCs (9, 10) and GABAergic IPSCs (8) in magnocellular neurons of the SON during lactation, although some controversy remains concerning whether these increases in glutamate and GABA inputs are accompanied by an increase in release probability.
Noradrenergic afferents make up about 10% of all synapses on magnocellular neurons (5), and norepinephrine plays an important role in the regulation of OT and VP secretion from SON neurons. It acts directly on magnocellular neuroendocrine cells to cause depolarization and spike firing (12), and it has recently been shown to facilitate the release of glutamate and attenuate GABA release onto SON and PVN magnocellular neurons (13, 14, 15, 16). Noradrenergic synapses participate in the structural plasticity of the magnocellular system during lactation, showing an increase in the number of synapses contacting OT neurons in the SON and PVN (5). Our previous studies demonstrated that, in untreated rats, norepinephrine increases magnocellular excitability by increasing presynaptic glutamate release (13, 14). The data presented here demonstrate that the noradrenergic modulation of both glutamate and GABA release is enhanced significantly after dehydration, showing an approximately 2-fold increase in the facilitation of glutamate release and the attenuation of GABA release. We were able to positively identify only relatively few cells immunohistochemically; however, similar changes in presynaptic and postsynaptic sensitivity to norepinephrine were seen in both the immunoidentified OT and VP neurons, suggesting that both cell types were implicated in the observed plasticity. The increased response to norepinephrine was disproportionate to the increase in spontaneous glutamate and GABA synaptic inputs: there was a doubling of the response to norepinephrine, compared with an approximately 30% increase in the frequency of EPSCs and IPSCs. This suggests either that there was a greater increase in norepinephrine receptor expression than in glutamate and GABA synapse numbers (i.e. that there was an increase in norepinephrine receptors per terminal) or that the noradrenergic receptor-effector coupling was enhanced with dehydration. To determine whether the increased responsiveness to norepinephrine both pre- and postsynaptically is caused by the selective plasticity of one or another of the -receptor subtypes responsible for the norepinephrine effects in magnocellular neurons (14, 15, 16) will require further study.
A recent study described interactions between presynaptic noradrenergic receptors and metabotropic glutamate receptors on presynaptic glutamate terminals, with 1-receptor activation blocking the inhibitory effect of metabotropic glutamate receptor activation on glutamate release in the PVN (27). Given that presynaptic metabotropic glutamate receptor signaling (26) and noradrenergic receptor signaling, as described here, are both up-regulated in chronically dehydrated rats, it will be interesting to determine whether these interactions are altered with chronic dehydration to promote a state of synaptic hyperexcitability.
Postsynaptic changes
After chronic dehydration, the whole-cell capacitance of recorded SON magnocellular neurons increased by about 20%, corresponding to a 20% increase in the surface area of the cells. Although smaller, this change in surface area is consistent with the somatic swelling (25–100% increase in somatic area) reported in morphometric studies of SON magnocellular neurons subjected to a similar dehydration regimen (18, 24). However, there was not any corresponding decrease in the input resistance of the magnocellular neurons, suggesting that the change in somatic surface with dehydration did not alter the electronic properties of the magnocellular neuronal membrane.
We recorded significant changes in the average amplitude and decay time of EPSCs, although not IPSCs, with chronic dehydration, implicating postsynaptic modifications specific to glutamatergic synapses. The 17% increase in EPSC amplitude suggests a postsynaptic receptor plasticity, that the synaptic glutamate receptor density or glutamate receptor-effector coupling increase with dehydration. An increase in ionotropic glutamate receptor density in SON magnocellular neurons has been reported after 2 d of dehydration using binding assays, although that change was limited to NMDA receptors (28). Other reports have also suggested that NMDA receptor subunits in magnocellular neurons undergo selective changes in numbers, both increases and decreases, during dehydration (29, 30). Another possibility is that the increase in EPSC amplitude reflects a presynaptic increase in synchronized multiquantal release of glutamate, as has been described under conditions of facilitated glutamate release (31). The reduction of the decay time of EPSCs observed after dehydration also suggests a postsynaptic change, either greater desensitization or faster deactivation of glutamate receptor channels in SON neurons from dehydrated animals, which could be caused by changes in either glutamate receptor subtype density or glutamate receptor composition. Interestingly, Stern et al. (10) reported the opposite effect of lactation on EPSC kinetics, showing an increase in the EPSC decay time at 8–14 d lactation. Similarly, Brussaard et al. (32) described a slowing of the IPSC decay with lactation, whereas we found no change in the amplitude or decay kinetics of IPSCs after chronic dehydration. These observations taken together suggest that changes in glutamate and GABA receptor expression may be different with different physiological stimuli. Further studies focused on changes in glutamate receptor expression and function with dehydration are required to determine the cause of the observed changes in postsynaptic responsiveness to synaptically released glutamate.
Norepinephrine elicited a tonic inward current in magnocellular neurons that was not blocked by ionotropic glutamate receptor antagonists, indicating that it was probably caused by activation of postsynaptic noradrenergic receptors. The norepinephrine-induced inward membrane current was significantly enhanced after chronic dehydration. Norepinephrine has been shown to elicit an inward current and a postsynaptic depolarizing response in magnocellular neurons from untreated rats (12, 14), and our findings suggest that this response is more robust in magnocellular neurons from dehydrated rats. This is consistent also with the increase in direct noradrenergic innervation of the magnocellular somata reported during lactation (5).
Functional significance
The frequencies of both EPSCs and IPSCs recorded in our experiments increased by about 30% (29 and 33%, respectively) after dehydration, which suggests that the glutamatergic and GABAergic innervation of magnocellular neurons increased nearly in parallel with neuronal surface area (
20% based on capacitance). One interpretation of the observed plasticity of synaptic innervation is that the increase in glutamate and GABA inputs represents a functional change in synaptic activity with dehydration that matches the change in cell size, thus providing a degree of synaptic regulation that is roughly consistent under the two conditions. This would imply that the synaptic changes that occur during dehydration are compensatory, designed to maintain the magnocellular neurons in a constant state of excitability through changing physiological conditions. Consistent with this interpretation is the fact that excitatory glutamatergic and inhibitory GABAergic synaptic inputs changed to the same degree, changes that would seem to cancel each other in terms of effects on postsynaptic excitability. However, the dramatic changes we found in the noradrenergic regulation of glutamate and GABA inputs to magnocellular neurons in dehydrated rats suggest another interpretation, that the increase in excitatory and inhibitory synaptic inputs creates a change in the activational potential of the magnocellular neurons and sets up a synaptic environment that promotes the cell type-specific electrical behaviors of these cells seen under different physiological conditions. That there was no reduction in the input resistance corresponding to the increased surface area of the magnocellular neurons tends to support the notion of the increased synaptic innervation conferring a greater capacity for synaptic activation (or inhibition) of these cells, rather than simple compensation for the increase in membrane surface area.
The changes in glutamatergic, GABAergic, and noradrenergic synaptic regulation of SON magnocellular neurons that occur with chronic dehydration are likely to have important functional consequences for the release of VP and/or OT. Although the parallel changes in glutamate and GABA synaptic inputs to magnocellular neurons with dehydration might not change the balance between excitation and inhibition in the SON, the increased noradrenergic regulation of these inputs, along with the enhanced postsynaptic effect of norepinephrine, would be expected to tip the balance significantly toward excitation. The interaction among these three different neurotransmitter systems may play a critical role not only in controlling the selective activation but also in determining the response characteristics (e.g. bursting) of VP and/or OT magnocellular neurons under different physiological conditions. This structural and functional plasticity of the magnocellular neuroendocrine system may serve as a model for similar forms of synaptic plasticity in other parts of the brain (33, 34).
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Acknowledgments |
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Footnotes |
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Abbreviations: aCSF, Artificial cerebral spinal fluid; AP5, L-2-amino-5-phosphonovalerate; DNQX, 5,6-dinitroquinoxaline-2,3-dione; EPSC, excitatory postsynaptic current; GABA, -aminobutyric acid; IPSC, inhibitory postsynaptic current; NMDA, N-methyl-D-aspartate; OT, oxytocin; PVN, paraventricular nucleus; SON, supraoptic nucleus; TTX, tetrodotoxin; VP, vasopressin.
Received June 2, 2004.
Accepted for publication July 29, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-receptor. Brain Res 365:192–197[CrossRef][Medline]
1-Adrenergic receptor activation depolarizes rat supraoptic neurosecretory neurons in vitro. Am J Physiol 251:R569–R574
2-adrenoceptors. Brain Res 807:61–69[CrossRef][Medline]
1 adrenoceptor-mediated inhibition of group III metabotropic glutamate receptors. J Neurosci 23:6223–6231This article has been cited by other articles:
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  P. Shi, S. D. Stocker, and G. M. Toney Organum vasculosum laminae terminalis contributes to increased sympathetic nerve activity induced by central hyperosmolality Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2007; 293(6): R2279 - R2289. [Abstract] [Full Text] [PDF]
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K. L. Freeman and V. L. Brooks AT1 and glutamatergic receptors in paraventricular nucleus support blood pressure during water deprivation Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2007; 292(4): R1675 - R1682. [Abstract] [Full Text] [PDF]
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 E. R. Gillard, C. G. Coburn, A. de Leon, E. P. Snissarenko, L. G. Bauce, Q. J. Pittman, B. Hou, and M. C. Curras-Collazo Vasopressin Autoreceptors and Nitric Oxide-Dependent Glutamate Release Are Required for Somatodendritic Vasopressin Release from Rat Magnocellular Neuroendocrine Cells Responding to Osmotic Stimuli Endocrinology, February 1, 2007; 148(2): 479 - 489. [Abstract] [Full Text] [PDF]
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J. G. Tasker, S. Di, and R. Malcher-Lopes Rapid Glucocorticoid Signaling via Membrane-Associated Receptors Endocrinology, December 1, 2006; 147(12): 5549 - 5556. [Abstract] [Full Text] [PDF]
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 |
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 G. R. J. Gordon and J. S. Bains Can homeostatic circuits learn and remember? J. Physiol., October 15, 2006; 576(2): 341 - 347. [Abstract] [Full Text] [PDF]
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 M. G. Kozoriz, J. B. Kuzmiski, M. Hirasawa, and Q. J. Pittman Galanin Modulates Neuronal and Synaptic Properties in the Rat Supraoptic Nucleus in a Use and State Dependent Manner J Neurophysiol, July 1, 2006; 96(1): 154 - 164. [Abstract] [Full Text] [PDF]
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 H. Schwimmer, L. Eli-Berchoer, and M. Horowitz Acclimatory-phase specificity of gene expression during the course of heat acclimation and superimposed hypohydration in the rat hypothalamus J Appl Physiol, June 1, 2006; 100(6): 1992 - 2003. [Abstract] [Full Text] [PDF]
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M. Grob and D. Mouginot Heterogeneous chloride homeostasis and GABA responses in the median preoptic nucleus of the rat J. Physiol., December 15, 2005; 569(3): 885 - 901. [Abstract] [Full Text] [PDF]
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S. Di, C. Boudaba, I. R. Popescu, F.-J. Weng, C. Harris, V. L. Marcheselli, N. G. Bazan, and J. G. Tasker Activity-dependent release and actions of endocannabinoids in the rat hypothalamic supraoptic nucleus J. Physiol., December 15, 2005; 569(3): 751 - 760. [Abstract] [Full Text] [PDF]
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