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1 Department of Psychiatry, University of Cincinnati, Cincinnati, OH, USA
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Abstract |
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(Received 19 October 2004;
accepted after revision 16 November 2004;
first published online 18 November 2004)
Corresponding author J. E. Stern: Department of Psychiatry, Genome Research Insitute, University of Cincinnati, 2170 E. Galbraith Rd, Cincinnati, OH 45237, USA. Email: javier.stern{at}psychiatry.uc.edu
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Introduction |
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NO is synthesized by NO synthases (NOS), which exist in three isoforms: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). nNOS has been found to be highly expressed in the SON and PVN (Arevalo et al. 1992), where it is colocalized with OT and VP neurones (Calka & Block, 1993; Miyagawa et al. 1994; Sanchez et al. 1994; Nylen et al. 2001). Furthermore, nNOS expression has been shown to be regulated in an activity-dependent manner during physiological or pathological states affecting fluid balance and reproductive functions. For example, nNOS mRNA expression in the SON and PVN is increased in response to osmotic stimuli (Kadowaki et al. 1994; Villar et al. 1994; Ueta et al. 1995) and after hypovolaemia (Ueta et al. 1998), whereas nNOS mRNA, as well as staining for nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-diaphorase, is down-regulated during late pregnancy and parturition (Okere & Higuchi, 1996; Srisawat et al. 2000). Based on these data, it has been proposed that within the magnocellular neurosecretory system (MNS), NO may function as an important inhibitory feedback regulator during conditions of high neuronal activity.
Besides the role of NO as an activity-dependent modulator during conditions of high hormonal demand, recent studies also support the presence of a functional endogenous basal NO tone (Goyer et al. 1994; Kadekaro & Summy-Long, 2000; Srisawat et al. 2000). Despite this physiological evidence, the cellular sources, targets and signals driving this basal NO production remain to be elucidated.
Due to the highly reactive nature and short half-life of NO, most studies have been limited to the analysis of NOS expression and localization. As a consequence, little is known about NO availability and production within specific cellular elements within the SON, or even whether nNOS-expressing neurones are indeed capable of producing NO. In the present study, we used a newly developed fluorescence method for the direct measurement of intracellular NO in magnocellular neurosecretory cells (MNCs) (Kojima et al. 1998). This approach has been efficiently used to localized NO sources in neuronal and non-neuronal tissue (Brown et al. 1999; Kashiwagi et al. 2002; Blute et al. 2003). Using this approach in combination with immunohistochemistry and electrophysiology, we aimed to elucidate the cellular sources and targets, functional relevance and mechanisms driving basal endogenous NO production within the MNS.
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Methods |
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Slice preparation
Coronal hypothalamic slices (150–300 µm) containing the SON were obtained using a vibroslicer (D.S.K. Microslicer, Ted Pella, Redding, CA, USA), as previously described (Stern et al. 1999). Briefly, rats were anaesthetized with pentobarbital sodium (50 mg kg–1 I.P.) and perfused through the heart with cold artificial cerebrospinal fluid (aCSF) in which NaCl was replaced by an equiosmolar amount of sucrose, a procedure known to improve the viability of neurones in adult brain slices (Aghajanian & Rasmussen, 1989). Following the perfusion, animals were rapidly decapitated and hypothalamic slices obtained as described above. The standard aCSF solution contained (mM): NaCl 120, KCl 2.5, NaH2PO4 1.25, MgSO4 1, CaCl2 2, NaHCO3 26, glucose 20 and ascorbic acid 0.4; pH 7.4 (297–300 mosmol l–1). After the slicing procedure, hypothalamic slices were placed in a holding chamber containing standard aCSF and stored at room temperature (22–24°C) until used.
Use and calibration of the nitric oxide (NO)-sensitive fluorescent indicator DAF-2
The NO-sensitive indicator DAF-2 was used to obtain information on intracellular NO availability. In the presence of NO and oxygen, the non-fluorescent DAF-2 is converted to the highly fluorescent DAF-2 triazole. Due to the extremely high sensitivity of this reaction (detection limit, 2–5 nM NO), and the fact that the intensity of the fluorescence signal generated is proportional to the amount of NO (Kojima et al. 1998; Suzuki et al. 2002), this approach permits the direct visualization, and semi-quantitative analysis of basal NO availability at the single cell level. To determine whether DAF-2-induced fluorescence in our system was also linearly related to NO availability, we initially used a cell-free system in which the emitted fluorescence from a series of solutions containing a fixed concentration of DAF-2 (10 µM) and increasing concentrations of the NO donor sodium nitroprusside (SNP; 0–1 mM) was measured. Images were aquired using a confocal microscope, and the intensity of the measured fluorescence (average of eight images per each solution containing varying concentrations of SNP), was plotted as a function of the SNP concentration. The concentration of DAF-2, as well as the imaging acquisition system, were the same as those used to measure DAF-2 fluorescence in the slice preparation (see below). As shown in Fig. 1, the addition of SNP resulted in a dose-dependent increase in the intensity of DAF-2-emitted fluorescence. The plot was best fitted with a linear regression function (R2 = 0.98).
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Following a 45–60 min preincubation in control aCSF, 150-µm hypothalamic slices were incubated in aCSF containing 10 µM DAF-2DA at room temperature, in the dark. Following a 30-min incubation period, slices were transferred to fresh aCSF and washed for at least 45 min in the dark. In some cases, each of the obtained slices from a rat brain was cut in half through a midline incision. While one-half followed the procedure described above, the other one was exposed (during the preincubation and DAF-2DA incubation periods), to either NOS antagonists 7-nitroindazole (100 µM), L-NAME (1 mM) and the NO scavenger 2-(4-carboxypheny)-4,4,5,5,-tetramethilimidazoline-1-oxyl-3-oxide (c-PTIO, 500 µM), or the glutamate receptor antagonists kynurenic acid (1 mM) or AP5 (100 µM). Following these procedures, slices were fixed in 4% paraformaldehyde overnight, subjected to immuhohistochemical procedures if needed (see below) and imaged using a scanning confocal microscope (see below).
Immunohistochemistry in DAF-2DA-loaded slices
To determine whether DAF-2 fluorescence (hereafter referred as NO-induced fluorescence, NOIF) was restricted to nNOS expressing neurones, hypothalamic slices loaded with DAF-2DA were fixed as described above, and then incubated for 24 h in the presence of a mouse monoclonal anti-nitric oxide synthase antibody (Sigma, St Louis, MI, USA; 1 : 400 dilution), followed by incubation for 2 h in the presence of a donkey anti-rabbit carboxy methyl indocynamine 5(Cy5)-labelled secondary antibody, used at a 1 : 400 dilution.
To determine whether differences in NOIF existed between OT and VP SON neurones, hypothalamic slices loaded with DAF-2DA were fixed and then incubated for 24 h in the presence of a cocktail of primary antibodies. To label VP neurones, one of the two following antibodies were used: a monoclonal mouse antibody raised against VP neurophysin (PS41, 1 : 1000 dilution, provided by Dr Harold Gainer, NIH), or a polyclonal rabbit anti-VP (Bachem, Torrence CA, USA; 1 : 100 000). Similar results were obtained with either of these antibodies. To label OT neurones, a polyclonal guinea pig anti-OT (Bachem; 1 : 100 000 dilution) was used. Reactions in primary antibodies were followed by incubation for 2 h in the presence of secondary antibodies (donkey anti-mouse or anti-rabbit Cy3-labelled and donkey anti-guinea pig Cy5-labelled secondary antibodies, all used at 1 : 400 dilution).
To determine whether glial cell bodies and processes also showed NOIF, DAF-2DA-loaded slices were incubated in the presence of a polyclonal rabbit anti-glial fibrillary acid protein (GFAP) (Chemicon International, Temecula CA, USA; 1 : 1000 dilution) followed by a 2-h incubation in the presence of a donkey anti-rabbit Cy5-labelled secondary antibody, 1 : 400 dilution). All antibodies were diluted with a phosphate-buffered saline (PBS) containing 0.1% Triton X-100. All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). All antibodies used have been previously characterized in detail. Control experiments in the present studies were performed by omitting primary or secondary antibodies. Following immunohistochemical reactions, slices were mounted and visualized using confocal microscopy (see below).
In a set of control experiments (n = 5), live brain slices (125 µm) were incubated in normal aCSF or in the presence of aCSF containing the NOS antagonist L-NAME (1 mM) and the NO scavenger c-PTIO (500 µM), for 30 min, and then fixed in 4% paraformaldehyde for 1 h. In two experiments, all solutions used contained the phosphodiesterase inhibitor 3-iso-butyl-1-methylxanthine (IBMX, 1 mM). Slices from these experiments were then subjected to a similar triple immunohistochemical procedure for cGMP, OT and VP. To this end, slices were incubated for 24 h in a mix of primary antibodies that included a polyclonal rabbit anti-cGMP (Chemicon International; 1 : 500 dilution), a monoclonal mouse antibody raised against VP neurophysin (PS41, 1 : 1000 dilution) and a polyclonal guinea pig anti-OT, as described above. This reaction was followed by a 2-h incubation in the presence of secondary antibodies (donkey anti-mouse Cy3-labelled, donkey anti-guinea pig Cy5-labelled and donkey anti-rabbit fluorescein isothiocyanate (FITC)-labelled secondary antibodies, all used at 1 : 400 dilution). All antibodies were diluted with PBS containing 0.1% Triton X-100.
Immunohistochemistry in thin hypothalamic sections obtained from fixed brains
Double and triple immunohistochemical fluorescence reactions were used to localize cGMP immunoreactivity (ir) in chemically identified SON neuronal perikarya, microvasculature, glial elements and nerve terminals. To label GABAergic and glutamatergic SON terminals, antibodies raised against glutamic acid decarboxylase 67 (GAD67), and the vesicular glutamate transporter (VGLUT2), respectively, were used. VGLUT is a reliable and specific marker of glutamate terminals (Takamori et al. 2000) and the VGLUT2 is the more abundant isoform found in the hypothalamus (Ziegler et al. 2002). For these studies, a group of rats (n = 8) were deeply anaesthetized with sodium pentobarbital (100 mg kg–1 I.P.) and perfused transcardially with 0.01 M PBS (150 ml) followed by 4% paraformaldehyde (500 ml). Brains were postfixed in 4% paraformaldehyde for 4 h at 4°C. Fixed brains were cryoprotected at 4°C with a 0.1 M PBS containing 30% sucrose for a minimum of 48 h. Sections (25 µm) were then cut using a cryostat, transferred into 0.01 M PBS and incubated in a solution of 0.01 M PBS with 0.01% Triton X-100 and 10% normal goat serum for 1 h.
For triple immunofluorescence reactions, sections were incubated for 24 h in a mix of primary antibodies that included a polyclonal rabbit anti-cGMP, a monoclonal mouse antibody raised against VP neurophysin, and a polyclonal guinea pig anti-OT, as described above. For double immunofluorescence reactions, sections were incubated for 24 h in a mix of primary antibodies that included the polyclonal rabbit anti-cGMP (as described above) in conjunction with one of the following antibodies: a monoclonal mouse anti-GAD67 (Chemicon International; 1 : 5000 dilution), a polyclonal guinea pig anti-VGLUT2 (Chemicon International; 1 : 5000 dilution), a polyclonal goat anti-GFAP (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1 : 1000 dilution) or a mouse monoclonal anti-nitric oxide synthase antibody (Sigma; 1 : 400 dilution). To localize cGMP in the SON microvasculature, the polyclonal rabbit anti-cGMP was combined with isolectin IB4 Alexa Fluor 568 conjugate, which has strong affinity for perivascular cells (Sahagun et al. 1989). Reactions in primary antibodies were followed by a 2-h incubation in secondary antibodies (donkey anti-rabbit FITC-labelled together with a donkey-Cy5-labelled raised against the respective species of the various primary antibodies used). All antibodies were diluted with PBS containing 0.1% Triton X-100. All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories Inc. Control experiments were performed by omitting primary or secondary antibodies.
Fluorescence confocal imaging acquisition and analysis
Hypothalamic slices or histological sections were examined with a Leica TCS SL confocal microscope (Leica Microsystems, Mannheim, Germany). DAF-2T (the fluorescent product of the reaction of DAF-2 with NO and O2), is readily fluorescent in the FITC light spectrum (Kojima et al. 1998). Confocal images of a single optical plane (1-µm thick) were obtained using appropriate emission and excitation wavelengths. The argon–krypton laser was used to excite the FITC and Cy3 fluorochromes, at 488 and 543 nm, respectively. The helium–neon laser was also used to excite the Cy5 fluorochrome at 633 nm. Fluorescence signal cross-talk among the different channels was avoided by setting image-acquisition parameters with individually labelled sections. Each optical section was averaged three times. In some cases, as noted throughout the text, six consecutive optical focal planes were obtained, and a projection image of the focal planes was displayed. Semi-quantitative analysis of NOIF fluorescence intensity was performed using imaging analysis software (Metamorph; Universal Imaging Corporation, Downingtown, PA, USA and Image Pro; Media Cybernetics, Silver Spring, MD, USA). Briefly, individual SON cells were manually traced, and the mean grey value contained within these regions was measured as an index of NOIF, and expressed as fluorescence arbitrary units (AUs), ranging from 0 (absolute black) to 255 (absolute green). Background fluorescence was substracted from all images. Neurones were arbitrarily considered to be positive for NOIF when the fluorescence intensity measured within the cell was twice that of the corresponding background level. When appropriate, SON neurones were also identified as either OT or VP, and the mean grey values of NOIF compared between the two cell populations. To determine colocalization of various fluorescent markers, confocal images obtained from the different fluorophores were merged. A positive colocalization was considered by the appearance of yellow (red + green) or white (blue + green) profiles in the merged image. Furthermore, an identifiable structure needed to be clearly discernible in each image prior to merging. The number of double-labelled neurones was estimated and expressed as percentage colocalization. A chi-square test was used to compare the incidence of double-labelled neurones.
A similar approach was used to determine the distribution of cGMPir within immunochemically identified SON neurones. However in this case, and due to the clustered nature of cGMPir, an automated tracing procedure incorporating a thresholding protocol (pixels were selected at 50% of the maximum fluorescence intensity) (Alvarez et al. 2004) was used to select cGMPir clusters inside manually traced OT or VP immunoreactive (OTir or VPir) neurones. The percentage of somatic area occupied by threshold cGMPir clusters was then calculated in each neurone. Neurones displaying a value 
10% of somatic area occupied by cGMPir were considered cGMP immunoreactive. A chi-square test was then used to compare the incidence of cGMP colocalization with OT and VP SON neurones.
To determine whether small cGMPir clusters located in close apposition to the surface membrane of SON neurones colocalized with, or were directly apposed to GAD67ir and/or VGLUT2ir boutons, individual SON neurones were imaged at high magnification (using a 63 x oil immersion objective, NA 1.32). As described above, selected regions around the perimeter of the cell were consecutively segmented first for the cGMPir signal (green channel), then for the GAD67 or VGLUT2 signals (blue channel). To determine colocalization, both channels were segmented simultaneously (Alvarez et al. 2004). The number of cGMP immunofluorescent punctae around the perimeter of individual neurones was measured, and the linear density (number of punctae (µm lineal membrane)–1) calculated. Percentage of colocalization was obtained from the number of immunostained profiles detected in both channels versus the number of profiles detected in each channel. Quantification of pixel intensity along line profiles constructed through boutons of interest was used to obtain a more detailed analysis of the spatial distribution of immunoreactive profiles within individual boutons, and to further confirm colocalization (Schneider & Lopez, 2002). Figures were composed using Adobe Photoshop (Adobe Systems Incorporated).
Electrophysiology
For electrophysiological recordings, a slice was transferred to a submersion-type recording chamber, continuously perfused (
2 ml min–1) with a standard solution bubbled with a gas mixture of 95% O2–5% CO2. All recordings were performed at 30–32°C. Patch pipettes (4–8 M
) were pulled from thin-walled (outer diameter, 1.5 mm; inner diameter, 1.17 mm) borosilicate glass (GC150T-7.5; Clark, Reading, UK) on a horizontal electrode puller (P-97; Sutter instruments, Novato, CA, USA). Gramicidin-based perforated patch recordings were obtained from SON neurones under visual control using an upright microscope (Axioscop, Zeiss, Germany) equipped with Nomarski infrared differential interface contrast (IR-DIC) optics and a water-immersion lens (40 x). This patch configuration was selected to prevent dialysis of intracellular signalling pathways potentially mediating NO effects. However, this prevented us, from immuno-identifying the phenotype of the recorded cell, as we have previously done using the whole cell configuration (Stern et al. 1999).
The pipette internal solution contained (mM): D-gluconic acid 130, KCl 20, Hepes 10, EGTA 0.5 and gramicidin 0.26. The pH was slowly tritrated to 7.25–7.30. The tip of the pipette was filled with a gramicidin-free solution. Electrical recordings were obtained using an Axopatch 200B (Axon Instruments, Foster City, CA, USA) patch-clamp amplifier. The series resistance was monitored throughout the experiment, and data were discarded if series resistance during recordings increased by two-fold compared to the series resistance obtained at the beginning of the recording. The voltage output was digitized at 16-bit resolution in conjunction with pClamp 8 software (Digidata 1320, Axon Instruments). Data were digitized at 10 kHz and transferred to a disk.
To study whether endogenously produced NO modulated the excitability of SON neurones, their firing activity was recorded in the current-clamp mode, and the effects of the relatively specific nNOS antagonist 7-nitroindazole (7-NI, 100 µM) (Moore et al. 1993; MacKenzie et al. 1994) alone, or in combination with the non-specific NOS inhibitor L-NAME (1 mM) and the NO scavenger c-PTIO (500 µM), were tested. Based on their initial firing pattern, neurones were classified as either continuously or phasically active (the latter being defined as those neurones displaying bursts of action potentials lasting at least 5 s and containing at least 20 action potentials, and with at least 5-s intervals between bursts (Brown et al. 1998).
In continuously firing neurones, mean basal values of firing rate (defined as the number of spikes s–1) were obtained by averaging these values over a 2-min period before drug applications. Due to the cell-to-cell variability in the latency of drug-induced responses (range, 133–218 s), the peak of the drug-induced response was determined, and the mean firing frequency was calculated during a period comprising 1 min before and 1 min after the peak effect. Similarly, values corresponding to the recovery from the drug treatment were calculated 5 min after the peak response.
In the case of phasically active neurones, the activity quotient, defined as the proportion of time in which a cell is active relative to the total recording time (Brown et al. 1998), was calculated and compared before and during drug applications. In these cases, longer periods of activity were recorded and analysed, to include several bursts of action potentials.
Solutions and drugs
All reagents were purchased from Sigma, except the NO scavenger 2-(4-carboxypheny)-4,4,5,5,-tetramethilimidazoline-1-oxyl-3-oxide (c-PTIO), and the NOS inhibitor 7-NI (both obtained from Alexis Biochemicals, Carlsbad, CA, USA).
Statistical analysis
Numerical data are presented as means ± S.E.M. Measurements of NOIF or cGMPir were obtained from individual neurones from different animals. As the inter-animal variability within each set of experiments was statistically insignificant (results not shown), data from a particular set of experiments were pooled together, and each magnocellular cell was treated as an independent variable, and the number of sampled cells used for statistical analysis. Analysis was performed in Statview (Abacus Concepts Inc, Berkeley, CA, USA), using the unpaired or paired non-parametric Mann Whitney U and Wilcoxon rank tests, respectively. Also, to compare differences in the incidence of an effect and/or proportions of double-labelled neurones, a chi-square test was used. Differences were considered statistically significant at P < 0.05.
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Results |
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To determine whether the NO-sensitive dye DAF-2 is an efficient indicator of basal NO production in the SON, hypothalamic brain slices containing the SON (obtained from a total of 24 male rats) were loaded with DAF-2DA, fixed, and examined under fluorescent light (see Methods). Examples of fluorescence confocal photomicrographs obtained from DAF-2-loaded slices containing the SON are shown in Fig. 2. High levels of basal NOIF were observed throughout the SON, which was clearly stronger than the fluorescence signal observed in the surrounding neuropil and in the perinuclear zone (PNZ) (Fig. 2A1). Within the SON, a highly heterogeneous distribution of NOIF was observed, with neurones showing either strong, weak or almost undetectable fluorescence signal.
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In a separate set of experiments (n = 5 animals, 133 neurones sampled), slices were exposed to a mix of NOS inhibitors (1 mM L-NAME and 0.5 mM 7-NI) and a NO scavenger (0.5 mM c-PTIO), before and during DAF-2DA loading. Images taken from these experiments showed a significant decrease of basal NOIF (control, 113.8 ± 2.8 AU; NO inhibitors, 48.6 ± 1.3 AU; n = 62 and 71 neurones in control and NOS inhibitors, respectively, P < 0.0001, Mann Whitney U test, Fig. 2B1 and 2B2). The lack of a complete block of basal NOIF by the NOS inhibitors, as also observed in previous studies (Brown et al. 1999; Kashiwagi et al. 2002; Blute et al. 2003; Pittner et al. 2003), could reflect incomplete NOS blockade and/or to the presence of non-enzymatic sources of NO (Zweier et al. 1999).
Besides neuronal elements, basal NOIF was also detected in glial processes (revealed by a positive GFAP immunoreactivity), as well as within the SON microvasculature, including arterioles and capillaries. Typical examples showing NOIF in glial and vascular elements are shown in Fig. 3. Similar to what we observed in neurones, the mix of NOS inhibitors decreased NOIF in glial and vascular cells (results not shown).
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NOIF and nNOS colocalize in individual SON neurones
Early studies have attempted to determine the cellular sources of NO in the MNS by localizing its synthesizing enzyme, nitric oxide synthase (NOS) (Arevalo et al. 1992; Calka & Block, 1993; Miyagawa et al. 1994; Sanchez et al. 1994; Nylen et al. 2001). However, a limitation of this approach is that measurements of NOS expression provide only an estimate of the potential of a cell to produce NO, and whether nNOS within MNCs constitutes an actual index of NO production is at present unknown. Thus, to assess the microtopographic relationship between the enzymatic neuronal source, and the actual production of NO, slices loaded with DAF-2DA were subsequently immunostained for nNOS (n
= 4 animals, 260 neurones sampled), and the number of double-labelled neurones was calculated. Typical photomicrographs showing simultaneous nNOS and NOIF staining are shown in Fig. 4. From a total of 260 NOIF-positive neurones sampled, 182 were found to be nNOS-positive (70%). Similarly, from a total of 198 nNOS-postive neurones sampled, 144 were found to be NOIF-positive (73%). Thus, while a high degree of double-labelled neurones was observed, our results indicate that 30% of SON neurones express either NOIF or nNOS immunoreactivity.
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To determine whether the heterogeneous distribution of basal NOIF observed in the SON was dependent on the neurochemical phenotype of SON neurones, slices loaded with DAF2-DA were subsequently immunostained with specific antibodies raised against OT and VP (see Methods, n
= 9 animals, 783 sampled neurones). The number of double-labelled neurones (e.g. NOIF and OT or VP), as well as NOIF intensity in OT and VP neurones was measured. Typical examples of hypothalamic slices labelled with DAF-2 and OT and VP immunostaning are shown in Fig. 5. From the total of NOIF sampled neurones, 59% (461/783) were VPir, and 30% (235/783) were OTir. This difference was statistically significant (P < 0.0001, chi-square test, Fig. 5B, left panel). Similarly, while 71% (368/518) of VPir neurones were NOIF-positive, only 42% (302/720) of OTir neurones were NOIF-positive (P < 0.0001, chi-square test, Fig. 5B, right panel). Finally, the overall intensity of NOIF in VP neurones was significantly higher than that observed in OT neurones (P < 0.02, Mann Whitney U test, Fig. 5C).
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Vasopressin neurones express higher levels of cGMP immunoreactivity than oxytocin neurones
A major mechanism underlying NO signalling in neurones involves the binding to, and activation of soluble guanylyl cyclase (sCG) (Knowles et al. 1989), with the subsequent production of cGMP. Thus, in an initial attempt to identify possible cellular targets of basal NO production, we used immuhohistochemical approaches to visualize cGMP in various cellular compartments within the SON.
A high degree of cGMPir was clearly observed in the SON, within neuronal, vascular, and glial elements, as well as diffusely throughout the neuropil (see Fig. 6). In SON neurones, cGMPir appeared punctate in nature, and was observed both intracytoplasmically, and in close proximity to the cell surface membrane (see Fig. 6A). cGMPir punctae had an average diameter of 0.6 ± 0.1 µm (n = 196). cGMP immunorectivity was also observed in GFAPir glial elements. In this case, and different from the clustered pattern observed in neurones, cGMPir was mostly diffuse. Examples of colocalization of cGMP and GFAP immunoreactivities are shown in Fig. 6B. It is interesting that cGMPir in GFAP-positive profiles was restricted to glial processes, with GFAP immunoreactive cell bodies lacking cGMPir. Diffuse cGMPir was also observed in isolectin-labelled vascular elements (examples shown in Fig. 6C).
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66% (122/185) were VP immunoreactive, and 35% (102/291) were OT immunoreactive. This difference was statistically significant (P < 0.0001, chi-square test, Fig. 5B, left panel).
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To determine whether the cGMPir observed in fixed brains represented NO-driven cGMP, a set of experiments (n = 5 animals, 260 neurones sampled) were performed in which living hypothalamic slices were incubated for 30 min in the presence of control aCSF, or aCSF containing the NOS antagonist or scavenger, L-NAME or c-PTIO, respectively, and then tested for cGMPir, VPir and OTir as above. In the absence of the phosphodiesterase inhibitor IBMX, little or no cGMPir was observed, probably due to a high rate of cGMP hydrolysis in living slices (see Fig. 8A). Experiments repeated in the presence of IBMX (n = 3) showed a more robust cGMPir (Fig. 8B). In this condition, and in agreement with our studies in fixed brains, the incidence of cGMPir was found to be significantly higher in VP when compared to OT neurones (32/42 sampled VP neurones (76%) and 21/50 sampled OT neurones (42%), P < 0.001, chi-square test). Furthermore, in the presence of L-NAME and c-PTIO, the mean somata area covered by cGMPir in immunoidentified VP neurones was significantly reduced from 14.7 ± 1.0% in control (n = 92 neurones) to 8.2 ± 1.0% (n = 76 neurones, P < 0.0001, Mann Whitney U test). The incidence of cGMPir in VP neurones was also significantly reduced from 64% (59/92) to 25% (19/76), respectively (P < 0.0001, chi-square test).
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Similar to the NOIF studies, we explored whether cGMPir observed in neuronal elements was limited to nNOS immunoreactive neurones. To this end, double-immunohistochemical studies combining cGMP and nNOS immunoreactivities were performed (n = 4 animals, 493 neurones sampled, Fig. 9). Immunoidentified cGMP and nNOS immunoreactive neurones, respectively, were sampled throughout the rostro-caudal extension of the SON, and the degree of colocalization between both immunoreactivities was determined. On average, we found 80.1 ± 3.5% of cGMP immunoreactive neurones to be also nNOSir, but only 47.5 ± 2.4% of nNOSir neurones displaying cGMPir. A relatively large group of nNOS immunoreactive neurones were also observed in the PNZ surrounding the SON. These neurones were clearly differentiated by their relatively smaller size compared to their neighbouring MNCs. Of interest, the majority of nNOS positive PNZ neurones lacked cGMPir (see Fig. 9C).
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To determine whether the small cGMPir clusters observed in close apposition to the surface membrane of SON neurones represented cGMP located in, or apposed to GABAergic or glutamatergic synaptic terminals, we performed double immunohistochemical studies combining primary antibodies raised against cGMP and the GABAergic or glutamatergic presynaptic markers glutamic acid decarboxylase (GAD67) and the vesicular glutamate transporter VGLUT2, respectively (n = 4 animals, Fig. 10A). The density of cGMPir clusters along the somatic perimeter of 36 randomly sampled SON neurones was calculated, and the proportion of those clusters that colocalized with either GAD67 or VGLUT2 immunoreactive profiles was estimated (n = 18 neurones in each case).
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Similarly to VGLUT2ir, abundant GAD67ir was found in the SON (Fig. 10A, right panel). GAD67ir was also punctate, and immunoreactive profiles appeared as small structures of varied sizes (diameter, 0.3–2.7 µm), surrounding neuronal profiles or in the neuropil. Cell body labelling in SON neurones was absent. In these neurones sampled, the density of cGMPir puncta along the cell membrane perimeter also averaged 0.4 ± 0.1 punctae µm–1. Co-localization of cGMPir and GAD67ir was observed in 13.2 ± 2.2% of sampled punctae. In this case, and in contrast to what was observed in cGMP/VGLUT2 profiles, cGMPir was found within the GAD67ir profiles. As shown in Fig. 10B (right panel), cGMP and GAD67 immunoreactivities, as well as the immunofluorescence intensity peaks along line profiles, clearly overlapped. A similar colocalization between cGMP and GAD67 was observed in the neuropil (Fig. 10C, right panel).
Basal NO tonically restrains ongoing firing discharge in phasic and continuously firing SON neurones
Our experiments measuring NOIF along with cGMP immunohistochemistry, suggest that NO is tonically produced and released within the SON, primarily from VP neurones. To determine whether this endogenous NO tone is functionally relevant, gramicidin-based perforated patch recordings were obtained from SON neurones
(n = 26) in a hypothalamic slice preparation, and the effects of NOS inhibitors on the firing activity of recorded neurones assessed. Recorded cells were classified based on their firing pattern in phasic or continuously firing cells. Phasic firing activity, a pattern characteristic of VP neurones (Poulain & Wakerley, 1982), was observed in 12 SON neurones, while a continuous, irregular activity was observed in the rest (n = 14). Following bath application of the nNOS inhibitor 7-NI (n = 4, 100 µM, 4 min), the activity quotient of phasically active neurones significantly and transiently increased from 0.33 ± 0.10 to 0.64 ± 0.2 (P < 0.05, Wilcoxon rank test), returning then to basal levels (0.25 ± 0.01, Fig. 11A). In separate experiments, phasically active neurones (n = 8) were exposed to a combination of 7-NI with the non-specific NOS blocker L-NAME (1 mM) and the NO scavenger c-PTIO (500 µM). Similar to the effects observed with 7-NI alone, the activity quotient transiently increased from 0.39 ± 0.05 to 0.65 ± 0.06 (P < 0.02, Wilcoxon rank test), returning then to basal levels (0.38 ± 0.05). No differences were observed between the percentage change in activity quotient induced by 7-NI alone, or in combination with the other inhibitors (P > 0.05, Mann Whitney U test, Fig. 11C). Neither of the NOS treatments used altered the mean intraburst firing frequency (control, 5.9 ± 0.8 Hz; combined NOS inhibitors, 6.0 ± 0.8 Hz; P > 0.05, Wilcoxon rank test).
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120 s). Glutamate receptor activation contributes to basal NO production in the SON
Glutamate NMDA receptors are known to be spatially coupled to nNOS (reviewed by Kiss & Vizi, 2001a). Furthermore, activation of NMDA receptors has been shown to induce NO-mediated responses in the SON (Amir, 1994; Bains & Ferguson, 1997). Thus, to determine whether basal NOIF in SON neurones is driven by activation of glutamate receptors, hypothalamic slices were preincubated with 100 µM AP5 (a specific NMDA receptor antagonist) or 1 mM kynurenic acid (a non-selective glutamate receptor antagonist) before and during DAF-2DA loading (n = 4 animals, 484 neurones sampled). Results from these experiments, in which NOIF was quantified in a total of 486 SON neurones, are summarized in Fig. 12. Blockade of NMDA receptors with AP5 significantly decreased NOIF in SON neurones by 20.2 ± 2.4% (control, 54.7 ± 0.9 AU; AP5, 44.5 ± 0.6 AU; P < 0.0001, Mann Whitney U test). It is interesting that a stronger inhibition in NOIF was observed when the non-selective glutamate receptor antagonist kynurenic acid was used (46.8 ± 4.7%, P < 0.05 as compared to percentage change in AP5; control, 99.3 ± 2.5 AU; kynurenic acid, 49.5 ± 0.6 AU; P < 0.0001, Mann Whitney U test, see Fig. 12C).
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Using a recently developed fluorometric method that enables the direct detection of intracellular NO within individual cells (Kojima et al. 1998; Brown et al. 1999; Blute et al. 2000; Tsumamoto et al. 2002; Blute et al. 2003; Li et al. 2003b; Rathel et al. 2003), in combination with immunohistochemistry and electrophysiology, we obtained novel information on the cellular sources, targets and functional relevance of tonic NO production within the MNS. To our knowledge, this is the first report to directly visualize intracellular NO availability in MNCs. Our results indicate that various cellular sources contribute to basal NO availability within the SON, which restrains ongoing firing activity of MNCs.
Fluorescence imaging of NO bioavailability as a tool to study NO production and cellular sources within the MNS
Because of the short half-life of NO and its highly reactive nature, it has been difficult to determine the local availability of the gas in vivo, especially at the cellular level. As a consequence, most studies so far have attempted to determine the cellular sources of NO by analysing the distribution of its synthesizing enzyme, nitric oxide synthase (NOS) (Arevalo et al. 1992; Calka & Block, 1993; Miyagawa et al. 1994; Sanchez et al. 1994; Nylen et al. 2001). However, a limitation of this approach is that measurements of NOS expression provide only an estimate of the potential of a cell to produce NO.
In an attempt to overcome this limitation, we have used in the present work the NO-sensitive fluorescent probe, DAF-2, which enables the direct visualization and semi-quantitative analysis of basal NO bioavailability at the single cell level. Original work by Nagano's group (Kojima et al. 1998; Suzuki et al. 2002), demonstrated that the fluorescence signal generated by DAF-2 was proportional to NO availability. Using a cell-free system and the NO donor SNP, we confirmed in this study that in our system, the intensity of NOIF signal generated was also linearly related to NO availability. Furthermore, the fact that the proportion of NOIF-labelled SON neurones in hypothalamic slices also increased proportionally to the amount of NO availability, along with the fact that NOIF was largely blunted in the presence of NO blockers/scavengers, indicates that DAF-2 constitutes an efficient and specific approach to measure NO bioavailability in the MNCs.
Heterogeneity in the cellular sources contributing to basal NO production within the SON
High levels of NO-induced fluorescence (NOIF) found within MNCs indicate that NO is actively produced under basal conditions in the SON. The high correlation between nNOSir and NOIF within individual cells (70%) suggests that nNOS is a major isoform contributing to SON basal NO production. Furthermore, our results showing a higher incidence and intensity of NOIF in VP, as compared to OT neurones, indicate that VP neurones constitute the major neuronal source of NO within the SON. However, these results do not necessarily rule out a role for basal NO in controlling OT secretion. In fact, inhibition of NOS activity in vivo increases secretion of both neurohormones (Kadekaro et al. 1997; Kadekaro & Summy-Long, 2000), and as shown in the present in vitro study, the electrical activity of both neuronal populations. Thus, while not contributing to basal NO production to the same degree as their VP counterparts, OT neurones may still be targeted by NO originating from alternative sources, including nearby VP neurones, vascular and/or glial sources. In fact, a role for non-neuronal NO sources in modulating OT neuronal activity is further supported by our electrophysiological results (see below). Alternatively, NO production by OT neurones may be dependent on active peripheral afferent inputs, likely to be absent in the slice preparation. Finally, it could be argued that cell-type differences in NOIF could in part be due to a differential dye loading and/or membrane esterase activity between OT and VP neurones. The fact that increasing NO levels by bath application of the NO donor SNP resulted in a significant increase in the proportion of OT neurones displaying NOIF argues against this possibility. Also, as the SON is known to contain only neurones that express either OT or VP (although under basal condition both peptides are co-expressed in a small proportion of them (Mezey & Kiss, 1991), the lack of immunoreactivity in 10% of neurones displaying NOIF most probably represents false negative immunoreactions, perhaps due to a lower tissue penetration of antibodies in relation to DAF-2DA.
The presence of NOIF in neurones lacking nNOSir, suggests that NO could arise from adjacent, nNOSir neurones. Alternatively, our results showing NOIF in glial and vascular cells suggest that these alternative cellular sources may also contribute to NO availability in SON neurones. Altogether, these results indicate that various cellular sources, including neurones, glial cells and the microvasculature, may contribute to basal NO availability in the SON.
Identification of NO receptive, cGMP-producing cellular elements within the SON
A major signal-transduction mechanism mediating NO actions within the central nervous system is the NO–cGMP pathway (Southam & Garthwaite, 1993). In fact, several reports support a role for this pathway underlying NO actions within the SON/VPN (Furuyama et al. 1993; Aguila, 1994; Melis & Argiolas, 1995; Briski, 1999; Yang & Hatton, 1999; Li et al. 2003a; Vacher et al. 2003) (but see Ozaki et al. 2000; Terrell et al. 2003). Within this context, we attempted to identify NO-receptive neurones in the SON by visualizing cGMPir. High levels of basal cGMPir were found in similar cellular elements that displayed strong NOIF, including MNCs, glial processes and the microvasculature. Furthermore, cGMPir was significantly higher in VP than in OT neurones, and similar proportions of NOIF and cGMPir were found to colocalize with nNOS-positive neurones. Thus, the high levels of cGMPir in SON somata, along with control experiments showing a decreased cGMPir in the presence of NOS blockers in live slices, suggest that basal NO actions in the MNS, involve, at least in part, direct cGMP-mediated effects. Of interest, these results are different from a recent study in mice, showing cGMPir limited to synaptic terminals and glial processes (Vacher et al. 2003).
In addition to directly modulating MNS excitability, several reports also indicate the presence of indirect, synaptically mediated effects (Bains & Ferguson, 1997; Ozaki et al. 2000; Stern & Ludwig, 2001). Thus, to determine whether cGMPir in the rat SON was also present in pre and/or postsynaptic compartments, we localized cGMPir in GABAergic and glutamatergic terminals, which together account for 70% of all synaptic contacts in the SON (El Majdoubi et al. 1997). Our results indicate that a proportion of SON GABAergic presynaptic terminals display cGMPir, providing an anatomical support for previous functional studies showing NO-mediated presynaptic modulatory actions on GABAergic activity in SON and PVN neurones (Bains & Ferguson, 1997; Ozaki et al. 2000; Stern & Ludwig, 2001; Li et al. 2003a,c).
In contrast to GABAergic terminals, cGMP clusters were found to appose, rather than to overlap glutamatergic terminals. Based on the well-characterized spatial coupling between NMDA receptors and nNOS (Kiss & Vizi, 2001b), these results would suggest a postsynaptic localization of cGMPir, representing perhaps NMDA-driven, NO–cGMP activation. In this sense, NMDA receptors mediate a large component of the glutamate synaptic response in SON neurones (Yang et al. 1994, 1995; Stern et al. 1999), and their activation triggers NO-mediated events (Bains & Ferguson, 1997). Additionally, NMDA-driven NO–cGMP activation is further supported by our results showing that glutamate receptor activation contributes to basally produced NO (see below). Analysis of cGMPir distribution at the ultrastructural level would be needed to confirm these observations.
Basally produced NO modulates ongoing firing activity of SON MNCs
Our electrophysiological studies further confirm the constitutive production of functionally relevant NO within the SON. In agreement with the higher levels of NOIF and cGMPir found in VP neurones, blockade of NO production with 7-NI, a relatively specific blocker of the nNOS isoform (Moore et al. 1993; MacKenzie et al. 1994), resulted in a stronger excitation in phasically active as compared to continuously active MNCs. While the expression of phasic and continuous firing patterns in vivo distinguishes VP from OT neurones, respectively (Poulain & Wakerley, 1982; Leng et al. 1991), the use of firing patterns to classify MNCs in vitro is more conflicting. While the expression of phasic activity in vitro is still a consistent feature found in the majority of VP neurones (Yamashita et al. 1983; Cobbett et al. 1986; Armstrong et al. 1994), continuous activity, though more often associated with OT neurones (Yamashita et al. 1987; Yang & Hatton, 1994), does not conclusively differentiate between the two (Armstrong, 1995). Thus, it is likely that in the present study, phasically active neurones represent VP neurones, while continuously firing neurones represent a mix of OT and non-phasic VP neurones.
The fact that the combined use of the non-specific NOS blockers/NO scavenger in phasic neurones evoked a similar excitation to that observed with 7-NI, suggests that these neurones are mostly targeted by NO originating from neuronal sources. A different pattern was observed in continuously firing neurones. In this case, a stronger effect was observed with the non-specific blockers, suggesting that non-neuronal sources of NO, such as endothelial and/or glial cells, may more efficiently target continuously firing MNCs.
While these studies strongly support a functional role for constitutive NO within the SON, they also raise interesting questions that deserve further investigation. Firstly, it is known that the spatial relationship between NO sources and their cellular targets is an important factor influencing NO specificity and efficiency (Paton et al. 2002). Thus, it will be important to determine whether the differential effect of NOS antagonists on phasic and continuosly firing neurones reflect a differential neuronal topographical organization in relation to vascular and/or glial elements. Secondly, as it is also known that NO can modulate the activity of MNCs through both direct and/or indirect mechanisms (Yang & Hatton, 1999; Ozaki et al. 2000; Stern & Ludwig, 2001), it will also be important to determine the relative contribution of such mechanisms to the actions of constitutive NO.
Basal NO production in the SON is partially driven by a glutamate-mediated mechanism
Activation of nNOS requires a rise in intracellular Ca2+, resulting in the production of NO through a Ca2+–calmodulin-dependent mechanism (Bredt & Snyder, 1990). Due to the high Ca2+ permeability and intimate spatial relationship with nNOS, NMDA receptors provide an efficient synaptically mediated route for Ca2+-dependent nNOS activation. In fact, the reduction in NOIF in MNCs observed in the presence of AP5 indicates that glutamate, acting in part through NMDA receptors, is an important signal driving basal NO production within the MNS. It is interesting that a larger reduction in NOIF was observed when a non-specific glutamate receptor antagonist was used, suggesting that non-NMDA receptors, most probably the AMPA subtype (Stern et al. 1999), also contribute to glutamate-mediated NOIF. We have previously shown that AMPA receptors in SON neurones are also Ca2+ permeable (Stern et al. 1999). Thus, their activation could also contribute to Ca2+-dependent NO production in MNCs.
SON astroglia as a potential source and target of NO
By modulating synaptic efficacy (Oliet et al. 2001), and by contributing to morphological and functional plasticity during conditions of high hormonal demand (Perlmutter et al. 1985; Theodosis & Poulain, 1999), SON glia play a fundamental role in controlling the MNS. While NO has been shown to mediate structural plasticity in other brain regions (Cogen & Cohen-Cory, 2000; Chen & Swanson, 2003; Park et al. 2004), whether it also plays a role in activity-dependent neuronal–glial remodeling in the SON remains to be elucidated.
The presence of NOIF and cGMPir in GFAP-positive processes within the SON suggests that the SON astroglia also contributes to NO production, and that they are targets of NO, respectively. Despite the absence of nNOSir in this cell population, it is likely that SON astroglia express the eNOS isoform (Gabbott & Bacon, 1996; Caillol et al. 2000). Alternatively, NO targeting the astroglia could arise from other sources, including neurones (nNOS) and/or endothelial cells (eNOS). Thus, the present results, along with a recent study by Vacher et al. (2003), suggest that NO may be an important molecule underlying interglial and/or neuronal–glial–vascular communication within the MNS.
In summary, results from this work support the presence of a functionally relevant constitutive NO tone within SON, and contribute to expand our current knowledge on the sources and targets of NO within the MNS. Future studies will be needed to further our understanding of the complex dynamic and bidirectional interactions among the various sources and targets of NO within this system, as well as NO interactions with other major neurotransmitter systems in the SON, including GABA and glutamate.
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Aguila MC (1994). Growth hormone-releasing factor increases somatostatin release and mRNA levels in the rat periventricular nucleus via nitric oxide by activation of
