Identification of the neuropeptide content of individual rat neurohypophysial terminals

Journal of Neuroscience Methods 163 (2007) 226–234

Identification of the neuropeptide content of individual rat neurohypophysial terminals Edward E. Custer 1 , Sonia Ortiz-Miranda 1 , Thomas K. Knott, Randi Rawson, Christian Elvey, Ryan H. Lee, Jos´e R. Lemos ∗ University of Massachusetts Medical School, Department of Physiology, 55 Lake Avenue North Worcester, MA 01655, United States Received 13 November 2006; received in revised form 1 March 2007; accepted 5 March 2007

Abstract The objective of this study was to develop a method that could reliably determine the arginine vasopressin (AVP) and/or oxytocin (OT) content of individual rat neurohypophysial terminals (NHT) ≥5 ␮m in diameter, the size used for electrophysiological recordings. We used a commercially available, highly sensitive enzyme-linked immunoassay (ELISA) kit with a sensitivity of 0.25 pg to AVP and of 1.0 pg to OT. The NHT content of AVP (2.21 ± 0.10 pg) was greater than OT (1.77 ± 0.08 pg) and increased with terminal size. AVP-positive terminals (10.2 ± 0.21 ␮m) were larger in diameter than OT-positive terminals (9.1 ± 0.24 ␮m). Immunocytochemical techniques indicated that a higher percentage (58%) of smaller terminals contained OT, and that a higher percentage (42%) of larger NHTs were colabeled. Similar percentages of AVP-positive terminals were obtained between immunocytochemical (73%) and ELISA (72%) methods when NHTs were assayed for AVP alone, but there was a higher percentage of OT terminals when using immunocytochemistry (43%) compared to ELISA (26%). The percent of AVP-positive (60%) and OTpositive (18%) terminals decreased when NHT were assayed for both AVP and OT. Therefore, the best method to reliably identify AVP-positive NHTs is to assay only for AVP, since this allows the conclusion that AVP-negative terminals contain only OT. © 2007 Elsevier B.V. All rights reserved. Keywords: AVP; OT; Posterior pituitary; ELISA; Immunocytochemistry

1. Introduction The hypothalamic-neurohypophysial system synthesizes arginine vasopressin (AVP) and oxytocin (OT), which are mostly secreted from nerve terminals in the neurohypophysis (Lemos, 2001). In these neurohypophysial terminals (NHT), calcium entry through pre-synaptic, high-threshold voltage-gated calcium channels induce neuropeptide release (Wang et al., 1993; Lemos, 2001). Four subtypes of high-threshold voltage-gated calcium channels have been identified in these terminals: L(Lemos and Nowycky, 1989), N- (Lemos and Nowycky, 1989; Wang et al., 1992), P/Q- (Wang et al., 1997) and R-types (Wang et al., 1999). The L- and N-type calcium channels are involved in the stimulated release of both AVP and OT, whereas, the Qand R-type calcium channels preferentially control AVP and OT release, respectively (Wang et al., 1997, 1999).

∗ 1

Corresponding author. Tel.: +1 508 856 8567; fax: +1 508 856 5997. E-mail address: [email protected] (J.R. Lemos). E.E. Custer and S. Ortiz-Miranda are co-first authors on this manuscript.

0165-0270/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2007.03.006

Other NHT population studies have also shown that stimulated release of neurohypophysial AVP and OT is differentially regulated. Purinergic receptor (P2X) activation elicits inward cationic currents (Knott et al., 2005) that preferentially stimulate AVP release (Troadec et al., 1998). In contrast, ␮-opioid receptor activation decreases depolarization induced OT release (Ortiz-Miranda et al., 2003) by preferentially inhibiting voltage dependent R-type calcium channels (Ortiz-Miranda et al., 2005). In order to accurately interpret any electrophysiological data and correlate it to peptide release studies, it is essential to be able to identify the neuropeptide content of individual NHTs. Therefore, we made use of an enzyme-linked immunoassay (ELISA, Assay Designs, Ann Arbor, MI) with adequate sensitivity to enable us to identify individual NHTs as containing AVP or OT subsequent to electrophysiological whole-cell recordings. A statistical description of isolated NHTs sizes and neuropeptide content is provided. Some of the results obtained with the ELISA were compared to those obtained using immunolabeling techniques as an independent corroborative measurement of the NHT population size and content.

E.E. Custer et al. / Journal of Neuroscience Methods 163 (2007) 226–234

2. Materials and methods 2.1. Isolation and collection of neurohypophysial nerve terminals Male Sprague–Dawley CD rats weighing 200–250 g (Taconic Farms, Germantown, NY) were sedated using CO2 and immediately decapitated, following the guidelines laid down by IACUCC, and the pituitary isolated (Cazalis et al., 1987). Following removal of the anterior and intermediate lobes of the pituitary, the neural lobe was dissociated in a buffer at 37 ◦ C containing (in mM): sucrose, 270; EGTA, 0.01; HEPES–Tris, 10, buffered at pH 7.25, with the osmolarity at 298–302 mOsmol/L. The dissociated neural lobe was plated into 35 mm polystyrene cell culture dishes (Corning, Corning, NY). After a waiting period of 2–3 min for the terminals to settle down on the bottom of the dish, the preparation was perfused at 37 ◦ C to remove any floating debris with a low free Ca2+ Locke’s saline containing (in mM): NaCl 140; KCl 5; EGTA 2; CaCl2 1.9 (estimated 3 ␮M free Ca2+ ); HEPES 10; glucose 10; MgCl2 1.2; pH 7.25, 298–302 mOsmol/L. This saline was replaced with a higher free Ca2+ (2.2 mM) Normal Locke’s solution following the initial rinse. Using an inverted microscope the NHTs were visually identified by their characteristic appearance, spherical shape, lack of nuclei, and relatively small size (5–12 ␮m in diameter). Sizes were recorded using a calibrated ocular micrometer (±0.25 ␮m). Thin borosilicate glass pipettes (Drummond Scientific Co., Broomall, PA) displaying resistances of 3–6 M containing either Normal Locke’s or a solution containing (in mM) Csglutamate 135; HEPES 10; glucose 5; CaCl2 2; MgCl2 1; tetraethylammonium (TEA) 20; amphotericin B (240 ␮g/mL dissolved in dimethyl sulfoxide; DMSO); pH 7.3 were placed against each individual terminal until a G seal resistance was obtained. At the end of each experiment, the content of the terminal was aspirated into the pipette with minimal extracellular fluid being aspirated (Wang et al., 1991). Some terminals were aspirated without undergoing a G seal formation. Once the terminals were aspirated, tips were broken inside a microcentrifuge tube and immediately frozen at −20 ◦ C until they were processed and assayed for AVP and/or OT. 2.2. Peptide identification A specific and sensitive enzyme-linked immunoassay (ELISA: Assay Designs, Inc.; Ann Arbor, MI) was used to determine the content of AVP and/or OT for individual terminals isolated and collected as described above. Assay sensitivity, which is the smallest amount of measurable AVP or OT that is reliably not zero, was determined in assay buffer supplied by the manufacturer. This was accomplished by running replicates (n = 8) of different AVP or OT concentrations and comparing those to the same number of replicates containing no hormone. The sensitivity of the AVP and OT ELISA’s in assay buffer was 0.25 and 1.0 pg/well, respectively. To determine if terminal isolation, collection, storage or preparation conditions affected the sensitivity of the AVP or OT ELISA, standard curves were

227

generated in Normal Locke’s solution, pipette solution, 0.1 and 0.5% Triton X-100 and standards prepared, frozen, and stored over night. Since none of the above conditions affected the AVP or OT standard curves (see Section 3) all subsequent standard curves for AVP or OT were prepared in the assay buffer supplied by the manufacturer of the ELISA. The detergent Triton X-100 (0.5%) was used to lyse the terminals and release AVP and/or OT into the assay buffer. When individual terminals were assayed for AVP (n = 226) or OT (n = 186) alone, the entire volume of buffer was assayed. Total volumes from individual terminals assayed for both AVP and OT (n = 197), were divided (100 ␮L was assayed for OT and 20 ␮L was assayed for AVP) based on the greater sensitivity of the AVP ELISA compared to the OT ELISA. This gave the best opportunity to detect both AVP and OT within an individual terminal. The cross reactivities of the AVP antibody with OT and the OT antibody with AVP were <0.001%. 2.3. Immunocytochemistry Dissociated isolated neurohypophysial terminals were obtained as described above. The homogenate was aliquoted equally (100 ␮L) onto two 22 mm × 22 mm glass microcoverslips placed in 35 mm polypropylene petri dishes, one coverslip for the IgG antibodies of the targeted peptides and one for the IgG control (see Section 2.6). One minute after plating, the dishes were filled with a 3 ␮M free calcium Locke’s solution and incubated at room temperature (RT) for 10 min, after which the low calcium Locke’s was replaced with Normal Locke’s for an additional 10 min. The isolated terminals were fixed with 4% paraformaldehyde in Normal Locke’s for 30 min, followed by a series of phosphate buffered saline (PBS) buffer rinses (3 × 10 min), then permeabilized in 1% Triton X-100 in PBS for 5 min. Antibodies raised against epitopes in neurophysin I or II, were used to identify the presence of OT and AVP, respectively, in each isolated terminal. Flourescently tagged secondary antibodies (Molecular Probes) were used to visualize and identify each terminal’s content. The oxytocin antibody was a mouse monoclonal antibody to neurophysin I generously provided by Dr. Harold Gainer (NIH), and the vasopressin antibody was an affinitypurified goat polyclonal antibody to neurophysin II (Santa Cruz Biotechnology, Santa Cruz, CA). Terminals were incubated for 1 h at RT with either their respective primary antibody (neurophysin I and II antibodies; diluted 1:100 and used at 2 ␮g/mL) or control peptides at identical concentrations, all in 1% Triton X-100 and 10% Donkey serum to block non-specific antibody binding. After a series of PBS buffer rinses (3 × 10 min) the terminals were incubated with either a 488 Fluorescein anti-goat (for vasopressin) or 594 Texas Red anti-mouse (for oxytocin) fluorescently tagged secondary antibody, raised in donkey (see Section 2.6), diluted 1:200 in 1% Triton X-100/PBS at RT for 1 h in the dark. After a final series of PBS rinses the coverslips were mounted on slides with the immuno-preservative Prolong (Molecular Probes, Eugene OR). Photographs of OT and AVP labeling were obtained with a Nikon Diaphot TMD microscope, using a Zeiss Plan-NEOFLUOR 100× oil immersion

228

E.E. Custer et al. / Journal of Neuroscience Methods 163 (2007) 226–234

lens, and fitted with a Photometrics SenSys CCD camera. Terminals were visualized using a Chroma 83500 filter cube excited with the appropriate filters using a Lambda DG4 high-speed filter changer (Sutter Instruments Incorporated, Novato, CA). Images were photographed at 10 ms exposures for brightfield photography and 800 ms exposures for fluorescently tagged neuropeptides, using Axon Imaging Workbench 2.1 software (Axon Instruments, Foster City, CA). 2.4. Stereological analysis The stereological analysis was performed with Stereo Investigator 4.33 (MicroBrightField, Colchester, VT; generously provided by Dr. Thomas Schoenfeld, University of Massachusetts Medical School) running on a Pentium III MHz computer (Dell, Austin, TX) under Windows 98 and 2000 (Microsoft, Redmond, WA). Color video images (color CCD camera, 640 × 480 pixels) of isolated neurohypophysial terminals on a Leica (Deerfield, IL) DMRE microscope were transmitted to the computer via a Flashpoint Intrigue video interface card (Integral Technologies, Indianapolis, IN) and displayed on a computer monitor (800 × 600 pixel resolution) in a real-time window. This resulted in an additional magnification of 22.8× over that achieved with each objective. Stereological sampling of each designated zone (5 mm × 5 mm) on the slide was controlled from Stereo Investigator via access to a motorized XY stage (MAC2000; Ludl Electronic Products, Hawthorne, NY). Terminals within each sampling area were identified as AVP and/or OT, according to their fluorescence and their sizes recorded. This software and hardware has been used to estimate olfactory cell populations, whose cell body diameters are smaller than that of terminals (Schoenfeld and Knott, 2004).

Fig. 1. Representative standard curves for arginine vasopressin and oxytocin. There is an increased sensitivity achieved with the AVP (open circles) compared to OT (solid circles) enzyme-linked immunoassay (ELISA). All points on the OT standard curve were shifted to the right compared to the AVP standard curve. B\Bo is the ratio of bound to free antigen (AVP or OT).

3. Results 3.1. Assay sensitivity and validation

Data are given as the mean ± S.E.M. Assay sensitivity of the AVP and OT ELISA was determined by Student’s t-test. The effect of terminal size (small, medium or large) on the mean content of AVP and OT was determined by ANOVA with subsequent Tukey’s HSD test to detect differences between means. The relationship between neuropeptide content (AVP or OT) and terminal diameter was determined by regression analysis. Statistical significance for all analyses was p < .05. All statistical analyses were accomplished using the computer-based program Sigma Stat (Jandel Scientific, San Rafael CA).

The sensitivities of the AVP and OT enzyme-immunoassay (ELISA) in assay buffer were 0.25 and 1.0 pg/well, respectively. These detection limits were determined by ANOVA, which established the lowest concentration of unlabeled hormone distinguishable from a sample containing no hormone. All points on the OT standard curve were shifted to the right compared to the AVP standard curve (Fig. 1), indicative of the decreased sensitivity of the OT assay. The addition of the detergent Triton X-100 (0.1 and 0.5%), overnight freezing, or use of the pipette solution (see Section 2) in which the neurohypophysial terminals were collected, had no effect on the AVP (Fig. 2) or OT (data not shown) standard curves. The cross-reactivity of the AVP and OT antibodies in assay buffer at 50% B/Bo , with OT and AVP, respectively, were <0.001% (Assay Designs, Ann Arbor, MI). Fig. 3 depicts the distribution of AVP and OT-positive terminals within typical AVP (Fig. 3, upper panel) and OT (Fig. 3, lower panel) standard curves. All terminals that tested positive for AVP were at 50–86% binding on the standard curve, whereas, >95% of terminals that tested positive for OT were at 85–92% binding, further indicating their different assay sensitivities and reliabilities.

2.6. Source of chemicals

3.2. Neuropeptide content of individual nerve terminals

All chemicals were obtained from Sigma–Aldrich (St. Louis, MO). The vasopressin (neurophysin II) antibody and control peptide were from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse IgG2b control (for OT) pre-immune antibody was purchased from Chemicon (Temecula, CA). The mouseoxytocin (neurophysin I) P38 antibody (Ben-Barak et al., 1985) was a kind gift from Dr. Harold Gainer (NIH). The Prolong and fluorecently tagged secondaries; donkey anti-mouse and donkey anti-goat were purchased from Molecular Probes (Eugene, OR).

A total of 609 neurohypophysial terminals, ranging in size from 5 to 16.5 ␮m in diameter, were assayed either for AVP (n = 226), or OT (n = 186) alone, or both AVP and OT (n = 197).

2.5. Statistical analysis

3.3. Assayed for AVP Seventy-three percent (164/226; Table 1) of terminals assayed for AVP alone were identified as AVP-positive. Furthermore, the percentage of AVP-positive terminals increased

E.E. Custer et al. / Journal of Neuroscience Methods 163 (2007) 226–234

Fig. 2. Assay conditions do not affect the arginine vasopressin standard curve. In the upper panel, AVP standard curves were generated by preparing standards in normal assay buffer or in the patch-pipette solution, in which the neurohypophysial terminals were collected. In the lower panel, AVP standard curves were generated by preparing standards in normal assay buffer (control), or in 0.1 and 0.5% Triton X-100, or standards prepared fresh and then stored frozen for 24 h prior to use. None of these conditions had any appreciable affect on the AVP or OT (data not shown) standard curves.

as terminal size increased (Table 2) and AVP-positive terminals were larger (p < .05; Table 3) than AVP-negative terminals. The mean AVP content per terminal was 2.26 ± 0.13 pg (n = 164; Table 1) and increased significantly (p < 0.05; Table 4 and Fig. 4A) as terminal size increased. 3.4. Assayed for OT Twenty-six percent (49/186; Table 1) of terminals assayed for only OT were identified as OT-positive. The percentage of OTpositive terminals increased (Table 2) as terminal size increased, but the mean diameter of OT-positive terminals was not different (p > 0.10; Table 3) from OT-negative terminals. The mean OT content per terminal was 1.60 ± 0.10 pg (n = 49; Table 1), but it did not increase (p > 0.10; Table 4 and Fig. 4B) as terminal size increased. 3.5. Assayed for AVP and OT When terminals were assayed for both AVP and OT, 49% (96/197) were AVP-positive, 7% (14/197) were OT-positive, 11% (21/197) were positive for both AVP and OT (i.e., colabeled) and 34% (66/197) were negative for both AVP and OT (Table 1). The percent of positive terminals, independent of

229

Fig. 3. The distribution of arginine vasopressin and oxytocin positive terminals within representative AVP and OT standard curves. In the upper panel, all terminals that tested positive for AVP (black circles) were between 50 and 86% binding on the standard curve (open circles). In the lower panel, >95% of terminals that tested positive for OT (black circles) were between 85 and 92% binding on the standard curve (open circles). This indicates that the neuropeptide content of the majority of terminals that tested positive for OT were at the upper limit of detection for the OT standard curve. Table 1 Neuropeptide content of individual neurohypophysial terminalsa determined by a specific enzyme immunoassay Assayed for

n

Percent

Mean neuropeptide content AVP (pg)

AVP Positive Negative

164 62

Total

226

OT Positive Negative

49 137

Total

186

AVP and OT AVP OT AVP and OT Negative

96 14 21 66

Total

197

AVP + (AVP and OT) OT + (AVP and OT)

117 35

72.56 27.44

OT (pg)

2.26 ± 0.13

1.60 ± 0.10

26.34 73.66

48.73 7.10 10.65 33.50

2.03 ± 0.16

59.39 17.76

2.13 ± 0.16 2.02 ± 0.14

2.58 ± 0.52

2.03 ± 0.24 2.01 ± 0.24

Individual NHT’s were aspirated into the recording pipette, transferred to a microcentifuge tube and frozen until neuropeptide content was determined by ELISA. a All neurohypophysial terminals had a diameter ≥5 ␮m.

230

E.E. Custer et al. / Journal of Neuroscience Methods 163 (2007) 226–234

Table 2 Enzyme immunoassay determination of AVP and/or OT content of small, medium and large neurohypophysial terminalsa Assayed for

n

Percent of terminals

Terminal size

AVP

OT

AVP and OT

Negative

AVP Medium Large

29 43

65.51 86.04

34.49 13.96

81.94

18.06

Total

72

OT Small Medium Large

25 94 63

20.00 24.46 30.15

80.00 75.54 69.85

Total

182

25.82

74.18

AVP and OT Small Medium Large Total

AVP + (AVP and OT)

OT + (AVP and OT)

42 78 58

14.28 51.28 62.06

11.90 2.56 10.34

7.14 12.82 10.34

66.68 33.34 17.26

21.42 64.10 72.40

19.04 15.38 20.68

178

46.06

7.30

10.67

35.97

56.73

17.97

Individual NHT’s were aspirated into the recording pipette, transferred to a microcentifuge tube and frozen until neuropeptide content was determined by ELISA. The data in this table is from a subset of terminals identified in Table 1 that had a size (small, medium or large) determination. a Small terminal diameter: ≥5 ␮m but <7 ␮m. Medium terminal diameter: ≥7 ␮m but <10 ␮m. Large terminal diameter: ≥10 ␮m.

neuropeptide content, increased as terminal size increased (Table 2). In addition, AVP-positive and colabeled terminals were larger (p < 0.05; Table 3) than negative terminals, whereas, OT-positive terminals were similar in size (p > 0.10; Table 3) to negative terminals. The mean AVP content per terminal was 2.13 ± 0.16 pg (n = 117; Table 1) and increased significantly (p < 0.05; Table 4 and Fig. 4A) as terminal size increased for both AVP-positive and colabeled terminals (Fig. 4C). The mean OT content per terminal was 2.02 ± 0.14 pg (n = 35; Table 1) Table 3 Relationship between terminal diameter and neuropeptide content for individual terminals identified with the enzyme immunoassay Assayed for

n

AVP Positive Negative

56 16

OT Positive Negative

47 135

AVP and OT AVP-positive OT-positive AVP and OT-positive Negative

65 12 15 63

Mean terminal diameter (␮m)

Percent

10.18 ± 0.21a 9.01 ± 0.43b

77.77 22.23

9.07 ± 0.24 8.68 ± 0.16

25.82 74.18

9.34 ± 0.21c 8.54 ± 0.73c,d 9.08 ± 0.41c 7.40 ± 0.23d

41.93 7.74 9.67 40.64

Individual NHT’s were aspirated into the recording pipette, transferred to a microcentifuge tube and frozen until neuropeptide content was determined by ELISA. The data in this table is from a subset of terminals identified in Table 1 that had a numerical size (␮m) determination. a,b Means within the AVP assay category with the same superscripts were not significantly different, whereas, means with different superscripts were significantly different at p < 0.05. c,d Means within the AVP & OT assay category labeled with the same superscripts were not significantly different, whereas, means with different superscripts were significantly different at p < 0.05.

but did not increase as terminal size increased for OT-positive (p > 0.10; Table 4 and Fig. 4B) or colabeled terminals (Fig. 4C). The mean AVP or OT content of colabeled terminals did not differ (p > 0.10; Table 1 and 4) from that of terminals that were only AVP- or OT-positive. 3.6. Immunohistochemical identification of neurohypophysial terminals Using a similar technique as described above, terminals were isolated and identified immunohistochemically (Fig. 5) as containing AVP (green), OT (red) or both AVP and OT (orange). Note that the red blood cell (RBC) in the bright field micrograph (Fig. 5, inset) does not label, indicative of the specificity of the AVP and OT antibodies. From a total of 3352 labeled terminals ranging in size from 1 to 17.5 ␮m in diameter, 45% (n = 1503) were AVP-positive, 50% (n = 1673) were OT-positive and 5% (n = 176) were positive for both AVP and OT (Table 5). AVP-positive terminals were larger (p < 0.05; Table 5) than OT-positive terminals and colabeled terminals (AVP and OT) were larger (p < 0.05; Table 5) than both AVP- and OT-positive terminals. Nearly 60% of terminals <5 ␮m in diameter were OTpositive. Within this size category, OT-positive terminals were smaller (p < 0.05; Table 5) than both AVP and colabeled terminals. Only 28% (n = 926; Table 5) of terminals identified immnunohistochemically, as AVP, OT or both AVP and OT were ≥5 ␮m in diameter. This is the size limitation for terminals used for electrophysiological recordings and aspirated via patch-pipettes for subsequent neuropeptide identification using the ELISA. For this population of terminals, 57% were AVPpositive, 28% were OT-positive and 15% were positive for both AVP and OT. When this terminal data is segregated into larger size categories, the general neuropeptide distribution shows that as terminal size increased the percent of OT-positive terminals

E.E. Custer et al. / Journal of Neuroscience Methods 163 (2007) 226–234

231

Table 4 Neuropeptide content of small, medium and large neurohypophysial terminalsa immuno-assayed for AVP, OT or AVP and OT Assayed for

n

Mean AVP (pg)

AVP Medium Large

19 37

1.72 ± 0.21b 2.77 ± 0.22c

Total

56

2.42 ± 0.17

OT Small Medium Large

5 23 19

1.77 ± 0.20 1.67 ± 0.11 1.59 ± 0.19

Total

47

1.65 ± 0.10

AVP and OT AVP-positive Small Medium Large

6 40 36

1.09 ± 0.19d 1.83 ± 0.21d 2.69 ± 0.29e

Total

82

2.16 ± 0.17

OT-positive Small Medium Large Total

5 2 6

1.89 ± 0.24 2.66 ± 1.35 2.02 ± 0. 39

13

2.07 ± 0.25

AVP and OT-positive Small 3 Medium 10 Large 6 Total

Mean OT (pg)

19

1.13 ± 0.28 2.67 ± 0.90 3.19 ± 0.96

1.74 ± 0.26 2.24 ± 0.30 1.85 ± 0.31

2.60 ± 0.56

2.05 ± 0.19

Individual NHT’s were aspirated into the recording pipette, transferred to a microcentifuge tube and frozen until neuropeptide content was determined by ELISA. The data in this table is from a subset of terminals identified in Table 1 that had a size (small, medium or large) determination. a Small terminal diameter: ≥5 ␮m but <7 ␮m. Medium terminal diameter: ≥7 ␮m but <10 ␮m. Large terminal diameter: ≥10 ␮m. b,c Means within the AVP assay category labeled with the same superscripts were not significantly different, whereas, means with different superscripts were significantly different at p < 0.05. d,e Means within the AVP & OT assay category for AVP-positive neurohypophysial terminals labeled with the same superscripts were not significantly different, whereas, means with different superscripts were significantly different at p < 0.05.

decreased, colabeled terminals increased and AVP-positive terminals did not change. 4. Discussion In this paper, we describe an effective method, an enzymelinked immunoassay, to determine the AVP and/or OT content of individual rat NHT’s ≥5 ␮m in diameter. This is the size limitation for terminals used in electrophysiological recording experiments (Lemos and Nordmann, 1986). The mean AVP (2.21 pg) and OT (1.77) content of individual rat NHT’s determined in the present study were similar to the neuropeptide content (1.76 pg) of rat NHTs estimated from the morphometric analysis of the neural lobe by Toescu and Morris (1990). Even though the mean NHT content of AVP and OT were similar, and

Fig. 4. Relationship between neuropeptide content and size of individual neurohypophysial terminals. (A) The amount of AVP per terminal (solid circles, n = 136) increased (p < 0.001, solid line) as terminal size increased in terminals assayed for only AVP or for AVP and OT. (B) The amount of OT per terminal (solid triangles, n = 74) did not change (p = 0.260, dashed line) as terminal size increased in terminals assayed for only OT or for AVP and OT. (C) In colabeled terminals, the amount of AVP per terminal (solid circles, n = 15) increased (p < 0.05, solid line) as terminal size increased, whereas, the amount OT per terminal (solid triangles, n = 15) was not affected by terminal size (p = 0.457). The solid (A and C) and dashed lines (B and C) represent regression lines generated using terminal diameter (␮m) as the independent variable and neuropeptide content (pg) as the dependent variable.

approximately 45–50% (Table 5) of the terminals contain either AVP or OT, the success and reliability of detecting AVP in individual rat NHTs was consistently greater than that for detecting OT. This seemed to be primarily due to the fact that the mean NHT content of AVP (2.21 pg) was considerably greater than the sensitivity (0.25 pg) of the AVP ELISA, whereas, the mean NHT content of OT (1.77 pg) was near the limit of detection (1 pg) of

232

E.E. Custer et al. / Journal of Neuroscience Methods 163 (2007) 226–234 Table 5 Relationship between neuropeptide content and terminal diameter (␮m) of individual rat neurohypophysial terminals identified immunohistochemically Size category neuropeptide n Diameter ≥1 ␮m AVP OT AVP and OT Total Diameter ≥1 ␮m<5 ␮m AVP OT AVP and OT Total Diameter ≥5 ␮m AVP OT AVP and OT

the OT ELISA. In support of this, approximately 16% of all AVP containing terminals had a neuropeptide content between 0.25 and 1 pg. If we assumed a similar trend for OT containing terminals, then all of those terminals would have been below the level of detection for the OT ELISA. In addition, the mean NHT content of AVP increased as terminal size increased, further enhancing our ability to detect AVP. This was not the case, however, for OT containing terminals. Another possibility is that the population of terminals assayed in the ELISA, which were ≥5 ␮m in diameter, has fewer OT than AVP containing terminals. In support of this, only 21% (396/1849) of OT containing terminals identified immunohistochemically were ≥5 ␮m in diameter, whereas, 40% (671/1679)

1503 1673 176

973 1418 35

530 255 141

AVP + (AVP and OT) OT + (AVP and OT)

671 396 210 52 97

Total

359

AVP + (AVP and OT) OT + (AVP and OT)

307 149

Total AVP + (AVP and OT) OT + (AVP and OT)

44.83 49.92 5.25

3.44 ± 0.02a 3.15 ± 0.16b 3.91 ± 0.11c

40.10 58.45 1.45

6.99 ± 0.08a 6.32 ± 0.09b 8.35 ± 0.17c

57.23 27.53 15.24

2426

926

Diameter ≥9 ␮m AVP OT AVP and OT

4.69 ± 0.05a 3.63 ± 0.03b 7.47 ± 0.19c

3352

Total

Diameter ≥7 ␮m AVP OT AVP and OT

Fig. 5. Fluorescent arginine vasopressin and oxytocin labeling in isolated neurohypophysial terminals. (A) Brightfield micrograph of a visual field containing representative isolated neurohypophysial terminals that were exposed to antibodies against AVP and OT (see panel B). Insert in panel A contains a brightfield image of a red blood cell (RBC) from a different visual field. (B) Double-labeling of the visual field in panel A with goat-anti-neurophysin II 1o antibody used to label AVP (1:100 dilution) visualized with a Fluorescein 488 2o donkey anti-goat antibody (1:200 dilution) and mouse anti-oxytocin 1o antibody (1:100 dilution) visualized with Texas Red 594 2o donkey anti-mouse antibody (1:200 dilution). Note colabeling of isolated terminal (orange) in upper left corner of panel B. Insert in panel B shows lack of double labeling, with the same antibodies, of the red blood cell in the panel A insert. Brightfield and fluorescent exposure times were 30 and 80 ms, respectively. Size bar = 10 ␮m.

Mean terminal diameter (␮m) Percent

84 14 70 168 154 84

72.47 42.77 8.87 ± 0.11a 8.61 ± 0.25a 9.52 ± 0.14b

58.50 14.48 27.02

85.52 41.50 10.52 ± 0.13 11.08 ± 0.53 10.11 ± 0.14

50.00 8.33 41.67 91.67 50.00

a,b,c Mean

terminal diameter within each size category labeled with the same superscripts were not significantly different, whereas, means with different superscripts were significantly different at p < 0.05.

of AVP containing terminals were ≥5 ␮m in diameter. In addition, within this size category, the percentage of OT-positive terminals decreased as terminal size increased. Furthermore, OT-positive terminals were smaller than both AVP-positive and colabeled terminals. This was also true for fractions of the bovine posterior pituitary in which the AVP/OT ratio increased from the lightest to the densest fractions indicating that AVP neurosecretosomes were larger than OT neurosecretosomes (Bindler et al., 1967). However, a recent report identified rat NHTs by the immunoblot method (Wang et al., 1991) and found that OTpositive and colabeled terminals were larger than AVP-positive terminals (OuYang et al., 2004). This discrepancy could be due to the method’s detection sensitivity and/or the limitation in the size of the terminals identified with the immunoblot method, even though this was similar to the size of terminals identified with the ELISA in the present study. The immunohistochemical identification of AVP (45%), OT (50%) and colabeled (5%) rat NHT’s, in this paper, was similar

E.E. Custer et al. / Journal of Neuroscience Methods 163 (2007) 226–234

to that reported for rat NHTs identified by electron microscopic immunohistochemistry (Mezey and Kiss, 1991) and was used to assess whether NHTs identified with the ELISA accurately depicted the population of terminals. When the entire content of a NHT was exclusively assayed for AVP using the ELISA, 73% of those terminals were identified as containing AVP. This was greater than the 57% of terminals ≥5 ␮m in diameter that were immunohistochemically identified as containing only AVP. This discrepancy can be explained by the fact that a portion of the AVP containing terminals identified with the ELISA would also contain OT and be classified as colabeled (AVP and OT) terminals. In support of this, when colabeled terminals were included in the total, 72% of NHTs ≥5 ␮m in diameter were immmunohistochemically identified as containing AVP, which is almost the same percentage identified as containing AVP with ELISA. This data supports the idea that a portion of the NHTs that were AVP-positive in the ELISA also contained OT (estimated to be 15%) and that few, if any, NHTs that were negative would actually contain AVP. This method, however, did not allow us to distinguish between colabeled terminals and terminals that contain only AVP. Furthermore, the close agreement in the percent of AVP containing NHTs between the immunohistochemical and the ELISA data when terminals were assayed for only AVP supports the idea that the 27% of terminals that were negative in the AVP ELISA should contain only OT. This is the percent of terminals ≥5 ␮m in diameter that were immunohistochemically identified as containing only OT. When the entire content of a NHT was assayed exclusively for OT, 27% of those terminals were identified as containing OT. This percent of OT-positive terminals would also include colabeled terminals. This was similar to the 28% of terminals ≥5 ␮m in diameter that were immunohistochemically identified as containing only OT, but considerably less than the 43% of terminals that were immunohistochemically identified as OTpositive when colabeled terminals were included in the total. This indicates that 37% of OT containing terminals were identified as negative when terminals were assayed only for OT. Since 15% of terminals ≥5 ␮m in diameter were immunohistochemically identified as colabeled terminals and again, no difference in content amount was seen, it is extremely likely that a significant portion of the 27% of OT containing terminals identified with the ELISA also contain AVP. In support of this, both immunohistochemically identified colabeled terminals and the percentage of positive terminals identified with the ELISA increased with terminal size. Therefore, the inability of the OT ELISA to accurately detect OT levels less than 1 pg coupled with the fact that OT terminals are smaller than both AVP and colabeled terminals are the most likely reasons for the discrepancy in the percent of OT containing terminals between the ELISA and the immunohistochemical data. Splitting the contents of a NHT and assaying for both AVP and OT with the ELISA enabled us to identify AVP (49%) and OT (7%) containing terminals, as well as, colabeled (11%) terminals. Interestingly, a greater percentage of colabeled terminals, which contain OT, were positively identified than terminals

233

that only contain OT. This was most likely due to the fact that colabeled terminals are larger than OT terminals and that the percentage of colabeled terminals increases as terminal size increases. Furthermore, splitting the terminal contents and assaying for both AVP and OT resulted in a decrease in the percent of terminals identified as containing only AVP or OT. When colabeled terminals were included in the total for AVP and OT, 60% were AVP-positive and 18% were OT-positive, which was less than the 72 and 27% that were positive when the entire content of a NHT was assayed for only AVP or OT, respectively. Furthermore, the percent of negatives was more pronounced for OT than for AVP (33 and 17%, respectively) most likely due to the decreased sensitivity of the OT ELISA. Attempts to compensate for this difference in sensitivity (dividing the total volume ∼80/20 in favor of OT) were not successful and did not significantly improve the ability to detect OT. This clearly indicates that splitting the contents of a NHT in order to assay for both AVP and OT decreases our ability to accurately detect both AVP and OT, with a more pronounced effect on our ability to identify OT containing terminals. However, we were able to identify 11% of assayed terminals as colabeled, which was similar to the 15% of terminals ≥5 ␮m in diameter that were immunohistochemically identified as colabeled. 5. In conclusion The close correlation between the immunohistochemical and ELISA data corroborated the information obtained with this latter method and provides a reliable approach to identify neuropeptide content in isolated neurohypophysial terminals. Due to the limitations of the OT ELISA, however, the most reliable approach to accurately identify neuropeptide content is by assaying the entire content of the neurohypophysial terminal for only AVP. This method, assaying only for AVP, accurately identified all AVP containing terminals, and any negative terminals as containing only OT. Thus, making it possible to identify NHT after patch-clamp recordings as being either vasopressinergic or oxytocinergic and, thus, correlate functional differences to neuropeptide content. Acknowledgements Contract grant sponsor: NIH; contract grant number: NS 29470 to J.R.L. References Ben-Barak Y, Russell JT, Whitnall MH, Ozato K, Gainer H. Neurophysin in the hypothalamo-neurohypophysial system. 1. Production and characterization of monoclonal antibodies. J Neurosci 1985;5:81–97. Bindler E, Labella FS, Sanwal M. Isolated nerve endings (neurosecretosomes) from the posterior pituitary. Partial separation of vasopressin and oxytocin and the isolation of microvesicles. J Cell Biol 1967;34(1):185– 205. Cazalis M, Dayanithi G, Nordmann JJ. Hormone release from isolated nerve endings of the rat neurohypophysis. J Physiol 1987;390:55–70. Knott TK, Velazquez-Marrero C, Lemos JR. ATP elicits inward currents in isolated vasopressinergic neurohypophysial terminals via P2X2 and P2X3 receptors. Pflugers Arch 2005;450(6):381–9.

234

E.E. Custer et al. / Journal of Neuroscience Methods 163 (2007) 226–234

Lemos JR. Magnocellular neurons. Encyc Life Sci 2001;A176:1–5, doi:10.1038/npg.els.000176. Lemos JR, Nordmann JJ. Ionic channels and hormone release from peptidergic nerve terminals. J Exp Biol 1986;24:53–72. Lemos JR, Nowycky MC. Two types of calcium channels coexist in peptidereleasing vertebrate nerve terminals. Neuron 1989;2:1419–26. Mezey E, Kiss JZ. Coexpression of vasopressin and oxytocin in hypothalamic supraoptic neurons of lactating rats. Endocrinology 1991;129(4):1814–20. Ortiz-Miranda SI, Dayanithi G, Coccia VJ, Custer E, Alphandery S, Mazuc E, et al. ␮-opioid receptor modulates peptide release from rat neurohypophysial terminals by inhibiting Ca2+ influx. J Neuroendocrinol 2003;15:888–94. Ortiz-Miranda SI, Dayanithi G, Custer E, Treistman SN, Lemos JR. ␮-Opioid receptor preferentially inhibits oxytocin release from neurohypophysial terminals by blocking R-type Ca2+ channels. J Neuroendocrinol 2005;17(9):583–90. OuYang W, Wang G, Hemmings Jr HC. Distinct rat neurohypophysial nerve terminal populations identified by size, electrophysiological properties and neuropeptide content. Brain Res 2004;1024:203–11. Schoenfeld TA, Knott TK. Evidence for the disproportionate mapping of olfactory airspace onto the main olfactory bulb of the hamster. J Comp Neurol 2004;476(2):186–201.

Toescu EC, Morris JF. Morphometric analysis of nerve endings isolated from bovine and rat neurohypophysis. J Anat 1990;3:1–17. Troadec JD, Thirion S, Nicaise G, Lemos JR, Dayanithi G. ATP-evoked increases in [Ca2+ ]i and peptide release from rat isolated neurohypophysial terminals via a P2X2 purinoceptor. J Physiol 1998;511:89–103. Wang G, Dayanithi G, Kim S, Hom D, Nadasdi L, Kristipati R, et al. Role of Q-type Ca2+ channels in vasopressin secretion from neurohypophysial terminals of the rat. J Physiol 1997;502:351–63. Wang G, Dayanithi G, Newcomb R, Lemos JR. An R-type Ca2+ current in neurohypophysial terminals preferentially regulates oxytocin secretion. J Neurosci 1999;19:9235–41. Wang X, Treistman SN, Lemos JR. Two types of high threshold calcium currents inhibited by ␻-conotoxin in nerve terminals of rat neurohypophysis. J Physiol 1992;445:181–99. Wang X, Treistman SN, Wilson A, Nordmann JJ, Lemos JR. Calcium channels and peptide release from neurosecretory terminals. NIPS 1993;8: 64–8. Wang XM, Treistman SN, Lemos JR. Direct identification of individual vasopressin-containing nerve terminals of the rat neurohypophysis after ’whole-cell’ patch-clamp recordings. Neurosci Lett 1991;11(1):125–8, 124.