Activity-Dependent Modulation of Neurotransmitter Innervation to Vasopressin Neurons of the Supraoptic Nucleus
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内分泌学杂志 2005年第1期
Department of Psychiatry (N.K.M., J.P.H.) and Cell Biology, Neurobiology, and Anatomy (J.P.H.), University of Cincinnati Medical Center, Cincinnati, Ohio 45237-0506; Department of Cell and Molecular Biology (S.D.), Tulane University, New Orleans, Louisiana 70118; and Department of Cell Biology and Neuroscience (C.M.P.), Montana State University, Bozeman, Montana 59717-3148
Address all correspondence and requests for reprints to: James P. Herman, Ph.D., Department of Psychiatry, University of Cincinnati, 2170 East Galbraith Road, Cincinnati, Ohio 45237-0506. E-mail james.herman@uc.edu.
Abstract
Confocal microscopy was used to assess activity-dependent neuroplasticity in neurotransmitter innervation of vasopressin immunoreactive magnocellular neurons in the supraoptic nucleus (SON). Vesicular glutamate transporter 2, glutamic acid decarboxylase, and dopamine ?-hydroxylase (DBH) synaptic boutons were visualized in apposition to vasopressin neurons in the SON. A decrease in DBH synaptic boutons per cell was seen upon salt loading, indicating diminished noradrenergic/adrenergic innervation. Loss of DBH appositions to vasopressin neurons was associated with a general loss of DBH immunoreactivity in the SON. In contrast, the number of vesicular glutamate transporter 2 synaptic boutons per neuron increased with salt loading, consistent with increased glutamatergic drive of magnocellular SON neurons. Salt loading also caused an increase in the total number of glutamic acid decarboxylase synaptic boutons on vasopressinergic neurons, suggesting enhanced inhibitory innervation as well. These studies indicate that synaptic plasticity compensates for increased secretory demand and may indeed underlie increased secretion, perhaps via neurotransmitter-specific, activity-related changes in synaptic contacts on vasopressinergic magnocellular neurons in the SON.
Introduction
THE MAGNOCELLULAR NEURONS of the supraoptic and paraventricular nuclei secrete the neurohypophysial hormones vasopressin and oxytocin, essential for osmotic balance, cardiovascular regulation, parturition, and lactation. After exposure to appropriate stimuli, e.g. osmotic loading (vasopressin) or suckling (oxytocin), hormone release is increased from neurohemal contacts in the neurohypophysis. Release is accompanied by pronounced increases in vasopressin and/or oxytocin biosynthesis in magnocellular cell somata (gene transcription, mRNA expression, and peptide content) (1, 2, 3, 4, 5, 6, 7).
Importantly, prolonged activation of magnocellular neurons causes marked neuroplastic changes in cellular morphology and innervation pattern. Enhanced biosynthetic activity is accompanied by increases in cytoplasmic and nuclear volumes as well as cross-sectional area (reviewed in Ref. 8). Dehydration increases cell size, the proportion of cells showing endoplasmic reticulum dilation, the proportion of neurosecretory granules with dense cores, and the number of multivesicular bodies in magnocellular neurons in the supraoptic nucleus (SON) (9). Osmotic stimulation also increases the number of magnocellular neurons showing multiple nucleoli (10). There is evidence for dendritic plasticity during lactation, in terms of both total length and new synapse formation (11, 12). Finally, lactation and dehydration elicit marked changes in glial morphology, with retraction of processes allowing enhanced neuron-neuron contacts and formation of gap junctions between magnocellular neurons (12, 13, 14).
Importantly, stimulation-induced changes in cell morphology are paralleled by altered neurotransmitter innervation; in the case of lactation, oxytocin and vasopressin neurons show increases in both excitatory and inhibitory synaptic contacts (13, 15). The neuroplastic changes seen in the oxytocinergic magnocellular system are not unique; parvocellular paraventricular nucleus (PVN) neurons show increased -aminobutyric acid (GABA) innervation after adrenalectomy or dexamethasone treatment (16). Therefore, it is clear that neuroplastic changes play a major role in cellular responses to repeated stimulation. In the past, anatomical studies of cellular innervation have required laborious and pain-staking electron microscopic analyses. In the current study, we employ an alternative approach (confocal microscopy) to quantify changes in excitatory and inhibitory neurotransmitter innervation of SON vasopressin neurons after chronic osmotic stimulation. Our results indicate that salt loading induces marked changes in glutamate, GABA, and noradrenergic/adrenergic innervation of vasopressin neurons and lend support to the hypothesis that altered neurotransmitter innervation may play a major role in enhanced neuronal excitability after chronic activation.
Materials and Methods
Dehydration procedure
Juvenile (4–5 wk old) male Sprague Dawley rats (Charles River, Wilmington, MA) were used in these experiments. Animals were housed on a 12-h light, 12-h dark cycle. Salt-loaded rats (n = 5) were given 2% NaCl in their drinking water and fed with dry pellet food ad libitum. They were killed after 8–10 d of salt loading. Control rats (n = 4) were age matched and housed under similar conditions with pure drinking water. Blood samples were collected before perfusion to measure osmolarity with a vapor pressure osmometer (model 5520; Wescor, Inc., Logan, UT). Rats were weighed before being killed. All animal protocols were approved by Tulane University Institutional Animal Care and Use Committee.
Preparation for immunocytochemistry
Rats received an overdose of sodium pentobarbital (50 mg/kg) and were subsequently perfused with 200 ml PBS at room temperature, followed by 250 ml 4% paraformaldehyde at room temperature. Brains were postfixed in paraformaldehyde for 12 h at room temperature and transferred to 30% sucrose in PBS at 4 C until the brains sank. Hypothalamic regions were cut at 30 μm on a sliding microtome (Microm, Walldorf, Germany) in a series of 12 and placed in cyroprotectant (0.1 M phosphate buffer, 30% sucrose, 1% polyvinylpyrrolidone, and 30% ethylene glycol). Cryoprotected sections were stored at –20 C until used for immunocytochemistry.
Dual immunocytochemistry
Sections were transferred from cryoprotectant to 50 mM potassium PBS (KPBS; 40 mM potassium phosphate dibasic, 10 mM potassium phosphate monobasic, and 0.9% sodium chloride) at room temperature. They were rinsed 4 x 10 min in KPBS and transferred to KPBS containing 0.3% H2O2 and incubated for 10 min. They were then rinsed 5 x 5 min in KPBS at room temperature and placed in blocking solution (50 mM KPBS, 0.1% BSA, and 0.2% Triton X-100) for 1 h at room temperature. Sections were then incubated in primary overnight at 4 C in blocking solution. Glutamic acid decarboxylase (GAD) primary antibody was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development, and maintained by The University of Iowa (Iowa City, IA); it was used at a 1:1000 dilution (17). Rabbit antivesicular glutamate transporter (VGlut) 2, antibody (Synaptic Systems, Gottingen, Germany) was directed against a recombinant glutathione-S-transferase-fusion protein containing amino acid residues 510–582 of rat VGlut2/differentiation-associated sodium-dependent inorganic phosphate cotransporter (DNPI), and was diluted 1:15,000. Mouse antidopamine ? hydroxylase (DBH) antibody was obtained from Chemicon (Temecula, CA) and diluted 1:25,000 (18). They were then rinsed 5 x 5 min in KPBS and incubated in biotinylated secondary antibody (Vector Laboratories, Inc., Burlingame, CA), diluted 1:500 in KPBS + 0.1% BSA for 1 h at room temperature. After 5 x 5 min KPBS rinses, sections were then treated with avidin-biotin complex (Vector Laboratories) 1:1000 in KPBS + 0.1% BSA for 1 h at room temperature, rinsed again in KPBS, and subsequently incubated in biotin-labeled tyramide (PerkinElmer Life Sciences, Inc., Boston, MA) 1:250 in KPBS with 0.3% H2O2 for 10 min at room temperature. After 5 x 5 min KPBS rinses, sections were incubated in Cy3-conjugated streptavidin (Jackson ImmunoResearch Labs, West Grove, PA) diluted 1:200 for 30 min at room temperature on a shaker.
For double labeling, sections were rinsed 5 x 5 min in KPBS and then incubated in the second primary antibody [(rabbit antivasopressin neurophysin (VP-NP) polyclonal antibody, supplied by Alan Robinson, University of California, Los Angeles], diluted 1:30,000, or SYN (rabbit antisynaptophysin antibody, Zymed, San Francisco, CA) diluted 1:300) in KPBS + 0.1% BSA at 4 C overnight and covered. After 5 x 5 min KPBS rinses, slices were then incubated in Alexa 488-labeled antirabbit antibody (Molecular Probes, Eugene, OR) diluted 1:200 in KPBS + 0.1% BSA at room temperature for 30 min and covered. Sections were rinsed 5 x 5 min in KPBS at room temperature after the final antibody incubation, mounted onto Superfrost Plus slides, and coverslipped with Gelvatol. To determine specificity of primary antibodies, control reactions were performed in the absence of one or both primary antibodies. In addition, VGlut2 immunofluorescence was specifically localized in regions known to express VGlut2 and not VGlut1 or VGlut3 neurons (e.g. thalamus) and did not correspond with patterns of staining seen using antibodies against VGlut1 (data not shown).
Image collection
Confocal imaging was performed on an LSM 510 microscope system (Zeiss, Thornwood, NY) in multichannel mode. All confocal images processed for analysis were collected using a x63 water immersion lens with a numerical aperture of 1.5; z-step, 0.5 μm; and image size 1024 x 1024 pixels. Cy3 was excited using the yellow line (568 nm) of the krypton/argon laser, whereas the green line (488 nm) was used to collect images of Alexa 488-labeled VP-NP or synaptophysin. At least four z-stacks of the supraoptic nucleus (0.5 μm thickness; 35–40 optical sections per animal) were collected at approximately –1.4 mm from bregma (19).
Image processing
All image processing was performed on an IBM (White Plains, NY) compatible computer using LSM 510 Image Browser software (Zeiss). Immunolabeled presynaptic boutons were examined for apposition to VP-NP-labeled magnocellular neurons. Boutons were considered to be in apposition only if there was no visible space between the bouton and the cell membrane in any optical section. Total numbers of boutons per neuron were counted across the entire z-axis for single neurons. This was accomplished by starting at the first (top) optical section containing a given neuron and counting the number of new boutons touching the cell surface through the entire thickness of the cell. To ensure that a bouton was not counted more than once, the images were examined by scanning back and forth through the z-axis several times. The total number of boutons in apposition was determined for each neuron examined. Four neurons per z-stack were counted and four z-stacks per animal were collected, resulting in a total of 16 neurons per animal. The cross-sectional area for each neuron examined was measured at the level of the nucleolus.
To determine whether the number of boutons varied by cell area with treatment group, we examined the bouton density. Bouton density was determined by counting the number of presynaptic boutons in apposition to the cell body in the optical section at the level of the nucleolus and dividing this value by the cross-sectional area.
Due to the paradoxical decrease in DBH innervation of arginine vasopressin (AVP) neurons, we sought to determine whether this loss was due to specific retraction of norepinephrine/epinephrine (NE/E) boutons from AVP neurons or a general loss of NE/E fibers in the SON. To assess this possibility, the density of DBH presynaptic boutons was determined in image stacks used for the colocalization analysis. The total density of boutons was estimated by taking the even images of a z-stack and exporting the red channel only. The image was then changed to gray scale and inverted in National Institutes of Health Image. Density scaling was used, and the area occupied by boutons was measured in a 170 x 170 pixel square. Two squares per image and 15–20 images per animal were measured, with the mean value for each animal used in statistical analyses.
Statistics
All data were analyzed by Student’s t tests. Significance was set at P < 0.05.
Results
Salt-loading significantly decreased body weight [t(7) = 76.292; P < 0.05] and increased plasma osmolality [t(7) = 11.117; P < 0.05] (Table 1). The results indicate that the salt-loading paradigm represented a substantial osmotic challenge.
TABLE 1. Body weight and plasma osmolality
As shown in Fig. 1, salt loading increased SON vasopressinergic cell cross-sectional area by about 58% [t(7) = 84.668; P < 0.05]. The observed cellular hypertrophy is consistent with previous results (10).
FIG. 1. Area of magnocellular somata. Somatic cross-sectional area was determined for vasopressin neurons in the SON of control and dehydrated rats, taken from the optical section midway through the nucleolus. *, Dehydration is significantly different from control at P < 0.05.
To determine whether our DBH, VGlut2, and glutamic acid decarboxylase (GAD)-immunoreactive structures label synaptic boutons, we double labeled brain sections with one of the neurotransmitter markers and synaptophysin. Fig. 2 illustrates the colocalization of DBH, VGlut2, or GAD with synaptophysin in the SON. Note that regions labeled by DBH alone were usually clearly separated from regions of synaptophysin alone, indicating that the label was in other parts of the DBH neuronal processes than the terminal. GAD and synaptophysin colocalized similar to what others have seen in area CA1 (20). The majority of VGlut2 labeling is colocalized with synaptophysin labeling. VGlut2 single label terminals were rare.
FIG. 2. Optical sections illustrating DBH (A–C; red), VGlut2 (D–F; red), and GAD (G–I; red) boutons colocalized with synaptophysin (green) immunoreactive presynaptic terminals.
To examine the effect of salt loading on glutamatergic, GABAergic, and noradrenergic/adrenergic innervation of SON vasopressinergic neurons, brain sections were labeled with DBH, VGlut2, or GAD and VP-NP. As expected, VGlut2, GAD, and DBH-immunoreactive boutons were seen in apposition to VP-NP-labeled magnocellular neurons in the SON. Figure 3 shows VP-NP immunoreactive neurons with DBH (Fig. 3, A and B), VGlut2 (Fig. 3, C and D), or GAD (Fig. 3, E and F) presynaptic terminals from control (Fig. 3, A, C, and E) and salt-loaded (Fig. 3, B, D, and F) rats. Note an apparent decrease in DBH appositions in salt-loaded animals, in contrast to the apparent increase in VGlut2 and GAD bouton contacts. Arrows illustrate examples of boutons that were counted in our analysis.
FIG. 3. Optical sections illustrating DBH (A and B; red), VGlut2 (C and D; red), and GAD (E and F; red) bouton appositions with vasopressin (green) immunoreactive neuronal somata in control (A, C, and E) and dehydrated (B, D, and F) SON neurons. Note the clear decrease in DBH appositions in salt-loaded animals, in contrast to the increase in VGlut2 and GAD bouton contacts. Arrows illustrate examples of boutons that were counted in our analysis. Magnification bars, 5 μM.
The total numbers of DBH, GAD, and VGlut2 presynaptic bouton appositions per vasopressinergic cell were determined. Salt loading decreased the total number of DBH presynaptic boutons per cell [t(7) = 6.4; P < 0.05] (Fig. 4A). A similar decrease in the density of DBH boutons [t(7) = 2.0; P < 0.05] was observed in salt-loaded rats (Fig. 4B).
FIG. 4. Analysis of DBH bouton counts per cell. Number of DBH boutons (A) decreased in salt-loaded animals relative to controls. Note that the decrease in number of boutons was not accompanied by a significant loss of overall DBH innervation of the SON region (P = 0.08) (B). *, Dehydration is significantly different from control, P < 0.05.
Salt loading resulted in a nearly 3-fold increase in the total number of VGlut2-immunoreactive boutons contacting VP-NP-positive neurons (Fig. 5B) [t(7) = 43.085; P < 0.05], consistent with a marked enhancement of glutamate innervation to this cell population. Notably, osmotic challenge also increased the total number of presynaptic GAD-positive boutons contacting VP-NP-immunoreactive SON neurons [t(7) = 24.326; P < 0.05] (Fig. 5A).
FIG. 5. Analysis of neurotransmitter bouton counts per cell. The number of GAD (A) and VGlut2 (B) boutons per cell increased in dehydrated rats. *, Dehydration is significantly different from control, P < 0.05.
Bouton density was examined to determine whether salt loading affected the density of neurotransmitter innervation (Fig. 6). In this analysis, the number of boutons was expressed as a ratio of total surface area, determined from optical sections containing the nucleolus of representative vasopressin neurons. Salt loading increased bothVGlut2 [t(7) = 26.849; P < 0.05] and GAD [t(7) = 10.632; P < 0.05] bouton density, whereas DBH [t(7) = 44.555; P < 0.05] bouton density was decreased. The data recapitulated the raw bouton counts, suggesting that salt loading affects both number and density of synaptic inputs. Bouton density was correlated with number of boutons per cell for DBH (correlation coefficient 0.968; P < 0.05) and VGlut2 (correlation coefficient 0.876; P < 0.05) but not for GAD (correlation coefficient 0.517; P = 0.16).
FIG. 6. Bouton density. The number of presynaptic VGlut2 (A), GAD (B), and DBH (C) boutons per cross-sectional area in VP-NP-labeled neurons in dehydrated and control-treated rats. Dehydration increased VGlut2 and GAD bouton density but decreased DBH bouton density. *, Dehydration is significantly different from control, P < 0.05.
Discussion
The results of this study support the hypothesis that magnocellular neurons show marked changes in glutamatergic, GABAergic and NE/E innervation as a consequence of prolonged stimulation. In the case of glutamate and GABA, both the number of boutons/cell and bouton density are increased, suggesting enhanced synaptic action of both transmitters on this cell population. In contrast, the number as well as density of DBH boutons are decreased with salt loading, consistent with a retraction of NE/E innervation from vasopressinergic neurons. Bouton density is highly correlated with number of boutons/cell for DBH and VGlut2 but not GAD.
The robust and readily apparent decrease in innervation of vasopressin neurons by DBH-positive boutons was unexpected. These data suggest a loss of direct noradrenergic/adrenergic innervation of vasopressin neurons, which is at odds with previous data showing enhanced responsiveness of magnocellular neurons to norepinephrine (21). However, it should be noted that a recent study described the excitatory effects of NE on magnocellular neurons of the SON, in part, via actions at presynaptic glutamatergic neurons (22). Thus, it is possible that withdrawal of DBH terminals from AVP cell bodies represents a functional rearrangement, allowing enhanced availability of NE/E to presynaptic terminals. It is important to note that the loss of boutons need not imply loss of NE signaling, as removal of neurotransmitter terminals may enhance sensitivity of the postsynaptic neurons to NE by up-regulation of adrenergic receptors.
Recent data suggest that the density of DBH innervation of the parvocellular subdivisions of the PVN is decreased after stimuli-provoking CRH release (23), suggesting stimulus-induced retraction of NE/E terminals in an analogous system of neuroendocrine neurons. As was the case with the magnocellular system after salt loading, the loss of innervation occurs despite increased responsiveness to NE release and enhanced responsiveness to adrenergic stimulation (23).
The apparent loss of NE/E innervation of magnocellular neurons after salt loading does not agree with electron microscopic studies performed in lactating rats, which demonstrate increased DBH contacts with oxytocin and vasopressin neurons (24). As such, it is possible that synaptic rearrangements may be differentially regulated in accordance with stimulus modality and/or retrograde signals emanating from vasopressin vs. oxytocin neurons. It should also be noted that the previous studies used adult rats; thus, it is possible that the discrepancies among the studies may be related to developmental differences in plasticity of neurotransmitter systems.
This study indicates that glutamatergic innervation of vasopressinergic neurons is markedly enhanced after chronic osmotic drive. Vasopressinergic neurons show more VGlut2-positive contacts. Previous studies indicate that vesicular glutamate transporters are specific to glutamate neurons (25). Three VGluts have been identified to date; of these, VGlut2 is the principal form identified in magnocellular regions of the PVN and SON (Refs. 26, 27, 28, 29 , Mueller, N. K., and J. P. Herman, unpublished observations). Thus, increased numbers of VGlut2 immunoreactive boutons likely reflect an enhanced capacity for glutamatergic stimulation. These data agree with electrophysiological studies demonstrating increases in glutamate miniature excitatory postsynaptic currents using this salt-loading model (21). Notably, prior electron microscopic studies indicate increased glutamatergic innervation of vasopressinergic neurons after stimulation of magnocellular neurons by suckling (30), suggesting that magnocellular neurons show similar changes in glutamatergic innervation after chronic activation by multiple stimuli. In addition, magnocellular neurons express both N-methyl-D-aspartate and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits (31, 32, 33, 34, 35, 36) and are directly responsive to glutamate and glutamatergic agonists (37, 38), demonstrating that glutamate is an essential stimulus for activation of the neurohypophysial system. Salt loading decreases NR2B protein levels (39) but increases NR1 protein levels in the SON (39, 40), suggesting an active modulation of glutamate receptors and, by inference, changes in glutamatergic activity.
Salt loading also increased both the total number of GAD-positive boutons per vasopressin cell and bouton density. Thus, there appears to be an increase in inhibitory as well as excitatory innervation of vasopressin neurons. These data parallel electron microscopic analyses showing enhanced GABA innervation of magnocellular neurons after lactation (41) and also agree with electrophysiological studies demonstrating enhanced excitatory and inhibitory postsynaptic currents after salt loading (21).
Despite the resolving power of confocal microscopy, our counting method is still limited to delineation of appositions. In the absence of electron microscopic analysis, we cannot conclude that these appositions are synapses. Nonetheless, it is clear that the optical sectioning method reveals stimulus-induced changes in glutamate and GABA appositions that are both predictable from previous morphological analyses (cf., Ref. 41) and in agreement with electrophysiological data (21). In addition, the method has permitted resolution of the novel finding that NE/E innervation of vasopressin neurons diminishes, rather then intensifies, after salt loading.
The current confocal strategy uses a method that identifies all immunoreactive boutons in apparent contact with vasopressin cells. The value of the technique lies in the ability to fully resolve the entire cell. In addition, individual boutons are typically present in 2–3 0.5 μm optical sections; as such, they are well within the resolving power of the confocal system and can be counted individually. However, the method does have some important caveats. First, counts are limited to neuronal cell somata because dendrites can be followed only within the thickness of a given section and can be obscured by immunoreactivity from neighboring neurons and processes. Second, cells need to be completely contained within the z-stack to obtain accurate bouton counts, thus limiting random selection to those neurons that meet this criterion. Finally, it remains possible that some subset of contacts is actually due to processes passing in near proximity to the labeled somata. Nonetheless, the current study indicates that confocal analysis using optical sectioning is a valuable tool with which to assess plasticity of neurotransmitter innervation to identified neurons after prolonged stimulation. This method can be readily adapted to analysis of other neurochemically defined systems and can provide valuable information on alterations in neurotransmitter capacity after stimulation, injury, or disease states.
Acknowledgments
We acknowledge Drs. Jeffrey Tasker and Robert Hennigan for their assistance and advice concerning these studies.
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Address all correspondence and requests for reprints to: James P. Herman, Ph.D., Department of Psychiatry, University of Cincinnati, 2170 East Galbraith Road, Cincinnati, Ohio 45237-0506. E-mail james.herman@uc.edu.
Abstract
Confocal microscopy was used to assess activity-dependent neuroplasticity in neurotransmitter innervation of vasopressin immunoreactive magnocellular neurons in the supraoptic nucleus (SON). Vesicular glutamate transporter 2, glutamic acid decarboxylase, and dopamine ?-hydroxylase (DBH) synaptic boutons were visualized in apposition to vasopressin neurons in the SON. A decrease in DBH synaptic boutons per cell was seen upon salt loading, indicating diminished noradrenergic/adrenergic innervation. Loss of DBH appositions to vasopressin neurons was associated with a general loss of DBH immunoreactivity in the SON. In contrast, the number of vesicular glutamate transporter 2 synaptic boutons per neuron increased with salt loading, consistent with increased glutamatergic drive of magnocellular SON neurons. Salt loading also caused an increase in the total number of glutamic acid decarboxylase synaptic boutons on vasopressinergic neurons, suggesting enhanced inhibitory innervation as well. These studies indicate that synaptic plasticity compensates for increased secretory demand and may indeed underlie increased secretion, perhaps via neurotransmitter-specific, activity-related changes in synaptic contacts on vasopressinergic magnocellular neurons in the SON.
Introduction
THE MAGNOCELLULAR NEURONS of the supraoptic and paraventricular nuclei secrete the neurohypophysial hormones vasopressin and oxytocin, essential for osmotic balance, cardiovascular regulation, parturition, and lactation. After exposure to appropriate stimuli, e.g. osmotic loading (vasopressin) or suckling (oxytocin), hormone release is increased from neurohemal contacts in the neurohypophysis. Release is accompanied by pronounced increases in vasopressin and/or oxytocin biosynthesis in magnocellular cell somata (gene transcription, mRNA expression, and peptide content) (1, 2, 3, 4, 5, 6, 7).
Importantly, prolonged activation of magnocellular neurons causes marked neuroplastic changes in cellular morphology and innervation pattern. Enhanced biosynthetic activity is accompanied by increases in cytoplasmic and nuclear volumes as well as cross-sectional area (reviewed in Ref. 8). Dehydration increases cell size, the proportion of cells showing endoplasmic reticulum dilation, the proportion of neurosecretory granules with dense cores, and the number of multivesicular bodies in magnocellular neurons in the supraoptic nucleus (SON) (9). Osmotic stimulation also increases the number of magnocellular neurons showing multiple nucleoli (10). There is evidence for dendritic plasticity during lactation, in terms of both total length and new synapse formation (11, 12). Finally, lactation and dehydration elicit marked changes in glial morphology, with retraction of processes allowing enhanced neuron-neuron contacts and formation of gap junctions between magnocellular neurons (12, 13, 14).
Importantly, stimulation-induced changes in cell morphology are paralleled by altered neurotransmitter innervation; in the case of lactation, oxytocin and vasopressin neurons show increases in both excitatory and inhibitory synaptic contacts (13, 15). The neuroplastic changes seen in the oxytocinergic magnocellular system are not unique; parvocellular paraventricular nucleus (PVN) neurons show increased -aminobutyric acid (GABA) innervation after adrenalectomy or dexamethasone treatment (16). Therefore, it is clear that neuroplastic changes play a major role in cellular responses to repeated stimulation. In the past, anatomical studies of cellular innervation have required laborious and pain-staking electron microscopic analyses. In the current study, we employ an alternative approach (confocal microscopy) to quantify changes in excitatory and inhibitory neurotransmitter innervation of SON vasopressin neurons after chronic osmotic stimulation. Our results indicate that salt loading induces marked changes in glutamate, GABA, and noradrenergic/adrenergic innervation of vasopressin neurons and lend support to the hypothesis that altered neurotransmitter innervation may play a major role in enhanced neuronal excitability after chronic activation.
Materials and Methods
Dehydration procedure
Juvenile (4–5 wk old) male Sprague Dawley rats (Charles River, Wilmington, MA) were used in these experiments. Animals were housed on a 12-h light, 12-h dark cycle. Salt-loaded rats (n = 5) were given 2% NaCl in their drinking water and fed with dry pellet food ad libitum. They were killed after 8–10 d of salt loading. Control rats (n = 4) were age matched and housed under similar conditions with pure drinking water. Blood samples were collected before perfusion to measure osmolarity with a vapor pressure osmometer (model 5520; Wescor, Inc., Logan, UT). Rats were weighed before being killed. All animal protocols were approved by Tulane University Institutional Animal Care and Use Committee.
Preparation for immunocytochemistry
Rats received an overdose of sodium pentobarbital (50 mg/kg) and were subsequently perfused with 200 ml PBS at room temperature, followed by 250 ml 4% paraformaldehyde at room temperature. Brains were postfixed in paraformaldehyde for 12 h at room temperature and transferred to 30% sucrose in PBS at 4 C until the brains sank. Hypothalamic regions were cut at 30 μm on a sliding microtome (Microm, Walldorf, Germany) in a series of 12 and placed in cyroprotectant (0.1 M phosphate buffer, 30% sucrose, 1% polyvinylpyrrolidone, and 30% ethylene glycol). Cryoprotected sections were stored at –20 C until used for immunocytochemistry.
Dual immunocytochemistry
Sections were transferred from cryoprotectant to 50 mM potassium PBS (KPBS; 40 mM potassium phosphate dibasic, 10 mM potassium phosphate monobasic, and 0.9% sodium chloride) at room temperature. They were rinsed 4 x 10 min in KPBS and transferred to KPBS containing 0.3% H2O2 and incubated for 10 min. They were then rinsed 5 x 5 min in KPBS at room temperature and placed in blocking solution (50 mM KPBS, 0.1% BSA, and 0.2% Triton X-100) for 1 h at room temperature. Sections were then incubated in primary overnight at 4 C in blocking solution. Glutamic acid decarboxylase (GAD) primary antibody was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development, and maintained by The University of Iowa (Iowa City, IA); it was used at a 1:1000 dilution (17). Rabbit antivesicular glutamate transporter (VGlut) 2, antibody (Synaptic Systems, Gottingen, Germany) was directed against a recombinant glutathione-S-transferase-fusion protein containing amino acid residues 510–582 of rat VGlut2/differentiation-associated sodium-dependent inorganic phosphate cotransporter (DNPI), and was diluted 1:15,000. Mouse antidopamine ? hydroxylase (DBH) antibody was obtained from Chemicon (Temecula, CA) and diluted 1:25,000 (18). They were then rinsed 5 x 5 min in KPBS and incubated in biotinylated secondary antibody (Vector Laboratories, Inc., Burlingame, CA), diluted 1:500 in KPBS + 0.1% BSA for 1 h at room temperature. After 5 x 5 min KPBS rinses, sections were then treated with avidin-biotin complex (Vector Laboratories) 1:1000 in KPBS + 0.1% BSA for 1 h at room temperature, rinsed again in KPBS, and subsequently incubated in biotin-labeled tyramide (PerkinElmer Life Sciences, Inc., Boston, MA) 1:250 in KPBS with 0.3% H2O2 for 10 min at room temperature. After 5 x 5 min KPBS rinses, sections were incubated in Cy3-conjugated streptavidin (Jackson ImmunoResearch Labs, West Grove, PA) diluted 1:200 for 30 min at room temperature on a shaker.
For double labeling, sections were rinsed 5 x 5 min in KPBS and then incubated in the second primary antibody [(rabbit antivasopressin neurophysin (VP-NP) polyclonal antibody, supplied by Alan Robinson, University of California, Los Angeles], diluted 1:30,000, or SYN (rabbit antisynaptophysin antibody, Zymed, San Francisco, CA) diluted 1:300) in KPBS + 0.1% BSA at 4 C overnight and covered. After 5 x 5 min KPBS rinses, slices were then incubated in Alexa 488-labeled antirabbit antibody (Molecular Probes, Eugene, OR) diluted 1:200 in KPBS + 0.1% BSA at room temperature for 30 min and covered. Sections were rinsed 5 x 5 min in KPBS at room temperature after the final antibody incubation, mounted onto Superfrost Plus slides, and coverslipped with Gelvatol. To determine specificity of primary antibodies, control reactions were performed in the absence of one or both primary antibodies. In addition, VGlut2 immunofluorescence was specifically localized in regions known to express VGlut2 and not VGlut1 or VGlut3 neurons (e.g. thalamus) and did not correspond with patterns of staining seen using antibodies against VGlut1 (data not shown).
Image collection
Confocal imaging was performed on an LSM 510 microscope system (Zeiss, Thornwood, NY) in multichannel mode. All confocal images processed for analysis were collected using a x63 water immersion lens with a numerical aperture of 1.5; z-step, 0.5 μm; and image size 1024 x 1024 pixels. Cy3 was excited using the yellow line (568 nm) of the krypton/argon laser, whereas the green line (488 nm) was used to collect images of Alexa 488-labeled VP-NP or synaptophysin. At least four z-stacks of the supraoptic nucleus (0.5 μm thickness; 35–40 optical sections per animal) were collected at approximately –1.4 mm from bregma (19).
Image processing
All image processing was performed on an IBM (White Plains, NY) compatible computer using LSM 510 Image Browser software (Zeiss). Immunolabeled presynaptic boutons were examined for apposition to VP-NP-labeled magnocellular neurons. Boutons were considered to be in apposition only if there was no visible space between the bouton and the cell membrane in any optical section. Total numbers of boutons per neuron were counted across the entire z-axis for single neurons. This was accomplished by starting at the first (top) optical section containing a given neuron and counting the number of new boutons touching the cell surface through the entire thickness of the cell. To ensure that a bouton was not counted more than once, the images were examined by scanning back and forth through the z-axis several times. The total number of boutons in apposition was determined for each neuron examined. Four neurons per z-stack were counted and four z-stacks per animal were collected, resulting in a total of 16 neurons per animal. The cross-sectional area for each neuron examined was measured at the level of the nucleolus.
To determine whether the number of boutons varied by cell area with treatment group, we examined the bouton density. Bouton density was determined by counting the number of presynaptic boutons in apposition to the cell body in the optical section at the level of the nucleolus and dividing this value by the cross-sectional area.
Due to the paradoxical decrease in DBH innervation of arginine vasopressin (AVP) neurons, we sought to determine whether this loss was due to specific retraction of norepinephrine/epinephrine (NE/E) boutons from AVP neurons or a general loss of NE/E fibers in the SON. To assess this possibility, the density of DBH presynaptic boutons was determined in image stacks used for the colocalization analysis. The total density of boutons was estimated by taking the even images of a z-stack and exporting the red channel only. The image was then changed to gray scale and inverted in National Institutes of Health Image. Density scaling was used, and the area occupied by boutons was measured in a 170 x 170 pixel square. Two squares per image and 15–20 images per animal were measured, with the mean value for each animal used in statistical analyses.
Statistics
All data were analyzed by Student’s t tests. Significance was set at P < 0.05.
Results
Salt-loading significantly decreased body weight [t(7) = 76.292; P < 0.05] and increased plasma osmolality [t(7) = 11.117; P < 0.05] (Table 1). The results indicate that the salt-loading paradigm represented a substantial osmotic challenge.
TABLE 1. Body weight and plasma osmolality
As shown in Fig. 1, salt loading increased SON vasopressinergic cell cross-sectional area by about 58% [t(7) = 84.668; P < 0.05]. The observed cellular hypertrophy is consistent with previous results (10).
FIG. 1. Area of magnocellular somata. Somatic cross-sectional area was determined for vasopressin neurons in the SON of control and dehydrated rats, taken from the optical section midway through the nucleolus. *, Dehydration is significantly different from control at P < 0.05.
To determine whether our DBH, VGlut2, and glutamic acid decarboxylase (GAD)-immunoreactive structures label synaptic boutons, we double labeled brain sections with one of the neurotransmitter markers and synaptophysin. Fig. 2 illustrates the colocalization of DBH, VGlut2, or GAD with synaptophysin in the SON. Note that regions labeled by DBH alone were usually clearly separated from regions of synaptophysin alone, indicating that the label was in other parts of the DBH neuronal processes than the terminal. GAD and synaptophysin colocalized similar to what others have seen in area CA1 (20). The majority of VGlut2 labeling is colocalized with synaptophysin labeling. VGlut2 single label terminals were rare.
FIG. 2. Optical sections illustrating DBH (A–C; red), VGlut2 (D–F; red), and GAD (G–I; red) boutons colocalized with synaptophysin (green) immunoreactive presynaptic terminals.
To examine the effect of salt loading on glutamatergic, GABAergic, and noradrenergic/adrenergic innervation of SON vasopressinergic neurons, brain sections were labeled with DBH, VGlut2, or GAD and VP-NP. As expected, VGlut2, GAD, and DBH-immunoreactive boutons were seen in apposition to VP-NP-labeled magnocellular neurons in the SON. Figure 3 shows VP-NP immunoreactive neurons with DBH (Fig. 3, A and B), VGlut2 (Fig. 3, C and D), or GAD (Fig. 3, E and F) presynaptic terminals from control (Fig. 3, A, C, and E) and salt-loaded (Fig. 3, B, D, and F) rats. Note an apparent decrease in DBH appositions in salt-loaded animals, in contrast to the apparent increase in VGlut2 and GAD bouton contacts. Arrows illustrate examples of boutons that were counted in our analysis.
FIG. 3. Optical sections illustrating DBH (A and B; red), VGlut2 (C and D; red), and GAD (E and F; red) bouton appositions with vasopressin (green) immunoreactive neuronal somata in control (A, C, and E) and dehydrated (B, D, and F) SON neurons. Note the clear decrease in DBH appositions in salt-loaded animals, in contrast to the increase in VGlut2 and GAD bouton contacts. Arrows illustrate examples of boutons that were counted in our analysis. Magnification bars, 5 μM.
The total numbers of DBH, GAD, and VGlut2 presynaptic bouton appositions per vasopressinergic cell were determined. Salt loading decreased the total number of DBH presynaptic boutons per cell [t(7) = 6.4; P < 0.05] (Fig. 4A). A similar decrease in the density of DBH boutons [t(7) = 2.0; P < 0.05] was observed in salt-loaded rats (Fig. 4B).
FIG. 4. Analysis of DBH bouton counts per cell. Number of DBH boutons (A) decreased in salt-loaded animals relative to controls. Note that the decrease in number of boutons was not accompanied by a significant loss of overall DBH innervation of the SON region (P = 0.08) (B). *, Dehydration is significantly different from control, P < 0.05.
Salt loading resulted in a nearly 3-fold increase in the total number of VGlut2-immunoreactive boutons contacting VP-NP-positive neurons (Fig. 5B) [t(7) = 43.085; P < 0.05], consistent with a marked enhancement of glutamate innervation to this cell population. Notably, osmotic challenge also increased the total number of presynaptic GAD-positive boutons contacting VP-NP-immunoreactive SON neurons [t(7) = 24.326; P < 0.05] (Fig. 5A).
FIG. 5. Analysis of neurotransmitter bouton counts per cell. The number of GAD (A) and VGlut2 (B) boutons per cell increased in dehydrated rats. *, Dehydration is significantly different from control, P < 0.05.
Bouton density was examined to determine whether salt loading affected the density of neurotransmitter innervation (Fig. 6). In this analysis, the number of boutons was expressed as a ratio of total surface area, determined from optical sections containing the nucleolus of representative vasopressin neurons. Salt loading increased bothVGlut2 [t(7) = 26.849; P < 0.05] and GAD [t(7) = 10.632; P < 0.05] bouton density, whereas DBH [t(7) = 44.555; P < 0.05] bouton density was decreased. The data recapitulated the raw bouton counts, suggesting that salt loading affects both number and density of synaptic inputs. Bouton density was correlated with number of boutons per cell for DBH (correlation coefficient 0.968; P < 0.05) and VGlut2 (correlation coefficient 0.876; P < 0.05) but not for GAD (correlation coefficient 0.517; P = 0.16).
FIG. 6. Bouton density. The number of presynaptic VGlut2 (A), GAD (B), and DBH (C) boutons per cross-sectional area in VP-NP-labeled neurons in dehydrated and control-treated rats. Dehydration increased VGlut2 and GAD bouton density but decreased DBH bouton density. *, Dehydration is significantly different from control, P < 0.05.
Discussion
The results of this study support the hypothesis that magnocellular neurons show marked changes in glutamatergic, GABAergic and NE/E innervation as a consequence of prolonged stimulation. In the case of glutamate and GABA, both the number of boutons/cell and bouton density are increased, suggesting enhanced synaptic action of both transmitters on this cell population. In contrast, the number as well as density of DBH boutons are decreased with salt loading, consistent with a retraction of NE/E innervation from vasopressinergic neurons. Bouton density is highly correlated with number of boutons/cell for DBH and VGlut2 but not GAD.
The robust and readily apparent decrease in innervation of vasopressin neurons by DBH-positive boutons was unexpected. These data suggest a loss of direct noradrenergic/adrenergic innervation of vasopressin neurons, which is at odds with previous data showing enhanced responsiveness of magnocellular neurons to norepinephrine (21). However, it should be noted that a recent study described the excitatory effects of NE on magnocellular neurons of the SON, in part, via actions at presynaptic glutamatergic neurons (22). Thus, it is possible that withdrawal of DBH terminals from AVP cell bodies represents a functional rearrangement, allowing enhanced availability of NE/E to presynaptic terminals. It is important to note that the loss of boutons need not imply loss of NE signaling, as removal of neurotransmitter terminals may enhance sensitivity of the postsynaptic neurons to NE by up-regulation of adrenergic receptors.
Recent data suggest that the density of DBH innervation of the parvocellular subdivisions of the PVN is decreased after stimuli-provoking CRH release (23), suggesting stimulus-induced retraction of NE/E terminals in an analogous system of neuroendocrine neurons. As was the case with the magnocellular system after salt loading, the loss of innervation occurs despite increased responsiveness to NE release and enhanced responsiveness to adrenergic stimulation (23).
The apparent loss of NE/E innervation of magnocellular neurons after salt loading does not agree with electron microscopic studies performed in lactating rats, which demonstrate increased DBH contacts with oxytocin and vasopressin neurons (24). As such, it is possible that synaptic rearrangements may be differentially regulated in accordance with stimulus modality and/or retrograde signals emanating from vasopressin vs. oxytocin neurons. It should also be noted that the previous studies used adult rats; thus, it is possible that the discrepancies among the studies may be related to developmental differences in plasticity of neurotransmitter systems.
This study indicates that glutamatergic innervation of vasopressinergic neurons is markedly enhanced after chronic osmotic drive. Vasopressinergic neurons show more VGlut2-positive contacts. Previous studies indicate that vesicular glutamate transporters are specific to glutamate neurons (25). Three VGluts have been identified to date; of these, VGlut2 is the principal form identified in magnocellular regions of the PVN and SON (Refs. 26, 27, 28, 29 , Mueller, N. K., and J. P. Herman, unpublished observations). Thus, increased numbers of VGlut2 immunoreactive boutons likely reflect an enhanced capacity for glutamatergic stimulation. These data agree with electrophysiological studies demonstrating increases in glutamate miniature excitatory postsynaptic currents using this salt-loading model (21). Notably, prior electron microscopic studies indicate increased glutamatergic innervation of vasopressinergic neurons after stimulation of magnocellular neurons by suckling (30), suggesting that magnocellular neurons show similar changes in glutamatergic innervation after chronic activation by multiple stimuli. In addition, magnocellular neurons express both N-methyl-D-aspartate and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunits (31, 32, 33, 34, 35, 36) and are directly responsive to glutamate and glutamatergic agonists (37, 38), demonstrating that glutamate is an essential stimulus for activation of the neurohypophysial system. Salt loading decreases NR2B protein levels (39) but increases NR1 protein levels in the SON (39, 40), suggesting an active modulation of glutamate receptors and, by inference, changes in glutamatergic activity.
Salt loading also increased both the total number of GAD-positive boutons per vasopressin cell and bouton density. Thus, there appears to be an increase in inhibitory as well as excitatory innervation of vasopressin neurons. These data parallel electron microscopic analyses showing enhanced GABA innervation of magnocellular neurons after lactation (41) and also agree with electrophysiological studies demonstrating enhanced excitatory and inhibitory postsynaptic currents after salt loading (21).
Despite the resolving power of confocal microscopy, our counting method is still limited to delineation of appositions. In the absence of electron microscopic analysis, we cannot conclude that these appositions are synapses. Nonetheless, it is clear that the optical sectioning method reveals stimulus-induced changes in glutamate and GABA appositions that are both predictable from previous morphological analyses (cf., Ref. 41) and in agreement with electrophysiological data (21). In addition, the method has permitted resolution of the novel finding that NE/E innervation of vasopressin neurons diminishes, rather then intensifies, after salt loading.
The current confocal strategy uses a method that identifies all immunoreactive boutons in apparent contact with vasopressin cells. The value of the technique lies in the ability to fully resolve the entire cell. In addition, individual boutons are typically present in 2–3 0.5 μm optical sections; as such, they are well within the resolving power of the confocal system and can be counted individually. However, the method does have some important caveats. First, counts are limited to neuronal cell somata because dendrites can be followed only within the thickness of a given section and can be obscured by immunoreactivity from neighboring neurons and processes. Second, cells need to be completely contained within the z-stack to obtain accurate bouton counts, thus limiting random selection to those neurons that meet this criterion. Finally, it remains possible that some subset of contacts is actually due to processes passing in near proximity to the labeled somata. Nonetheless, the current study indicates that confocal analysis using optical sectioning is a valuable tool with which to assess plasticity of neurotransmitter innervation to identified neurons after prolonged stimulation. This method can be readily adapted to analysis of other neurochemically defined systems and can provide valuable information on alterations in neurotransmitter capacity after stimulation, injury, or disease states.
Acknowledgments
We acknowledge Drs. Jeffrey Tasker and Robert Hennigan for their assistance and advice concerning these studies.
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