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Morphometric changes of specific located vasopressin-reacting parvicellular neurons in the paraventricular nucleus of the rat after adrenalectomy
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Neuropeptides (1990) 17,127-134 0 Longman Group UK Ltd 1990
Morphometric Changes of Specific Located Vasopressin-Reacting Parvicellular Neurons Paraventricular Nucleus of the Rat After Adrenalectomy F. SANCHEZ*, J. CARRETERO”, F. SANCHEZ-FRANCOt, J. A. JUANES” and R. VAZQUEZ”
J. M. RIESCO’,
in the
E. BLANCO”,
*Department of Human Anatomy and Histology. Avda Campo Charro s/n. Faculty of Medicine. University of Salamanca, 37007 Salamanca, Spain. tEndocrinology Service of the Ramdn y Cajal Hospital, Madrid. (Reprint request to FSl
Abstract-The morphological-morphometric consequences of bilateral adrenalectomy on vasopressin-reacting neurons of the paraventricular nucleus of the rat hypothalamus were analyzed. Bilateral adrenalectomy led to a dramatic increase in the cellular area as well as the number of immunoreactive ceils (when compared to those obtained in normal colchicinetreated animals) in the neurons located in the anterior, medial and periventricular parvicellular subdivisions of the paraventricular nucleus. By contrast, no changes were observed in either the dorsal or lateral parvicellular subdivisions or in any of the magnocellular subdivisions of the paraventricular nucleus.
Introduction In recent years various peptides have been implicated in the hypothalamic control of corticotropic hormone (ACTH) secretion; the ACTH releasing factor (CRF) and vasopressin (VP) have received particular attention (4, 8, 25, 27), although other peptides, such as VIP (9) are gradually being implicated in this process. Different papers, including studies from our laboratory, have highlighted the important role of Date received 9 March 1990 Date accepted 28 May 1990
VP-producing neurons in the parvicellular regions of the hypothalamic paraventricular nucleus (PVN) in the secretory control of pituitary ACTH release (5, 12, 14, 19, 26). These parvicellular neurons do not normally express VP immunoreactivity (20). Morphometry has been shown to be an adequate technique to valorate the consequence of different experimental states on the central nervous system. Accordingly, in this study the poseffects of bilateral sible morphometric adrenalectomy on VP-producing neurons of the PVN were evaluated in an immunocytochemicalmorphological-morphometric study. 127
128 Material and Methods
The animals were housed in standard laboratory conditions: seasonal light-dark cycle, temperature 22 + 2°C R.H. 50 + 5%, and food and water ad libitum. Thirty male and female Sprague-Dawley rats were used, divided into three groups: 1) Normal animals (n = 10,5 per sex); 2) Animals pretreated with 90 t~,gof colchicine dissolved in 15 ~1 of saline and administered in the lateral ventricle by stereotaxic surgery 24h prior to sacrifice (n = 10, 5 per sex); 3) Bilaterally adrenalectomized animals (15 days) under ketamine anaesthesia (n = 10, 5 per sex). Like those of group 2, the rats were pretreated with colchicine (24h prior to sacrifice). Following adrenalectomy (dorsal approach), the animals’ drinking water was supplemented with sodium chloride (0.9%) and sucrose (5%). The completeness of adrenalectomy was verifed by necropsy. After sacrifice by decapitation, the hypothalamic-hypophyseal block was fixed by immersion in Bouin-Hollande solution and embedded in paraffin. Serial 5 pm frontal sections were obtained with a microtome. fmmunocytochemzktry
After inhibiting endogenous peroxidase by Passing the samples through a methanol bath Hz02 (22), the sections were pretreated with normal Swine serum (DAKO). Following this, the PAP immunocytochemical method was applied (21). As primary serum, anti-VP Serum was employed (10, 17) at a dilution of l/1000. The rest of the sera (anti-rabbit immunoglobulin swine serum and PAP soluble complex) were supplied by DAK0 (see 14). The reaction was visualized using a 0.05% solution of 3-3’ diaminobenzizidine in TRIS-HCL buffer containing 0.01% hydrogen peroxide. Preabsortion test with VP (SIGMA) and substitution test of the primary serum by normal rabbit serum were carried out. After preabsortion or substitution test the immunoreaction was completely abolished.
NEUROPEPTIDES
(never more than three per subdivision and section) in both the parvicellular and magnocellular subdivisions (100 cells in each subdivision, per sex and different experimental group of animals were analyzed, with the exception of the parvicellular subdivisions of group 1 in which, with the technique employed, the VP-reactive neurons were very scarce and presented a very weak reaction intensity). B) Number of reacting cells. The number of cells was calculated by analyzing each subdivision, sex and group of animals, with the exception of the parvicellular subdivisions of group 1 (given the scarcity and the weakness of immunoreaction of the VP-reacting neurons in the parvicellular subdivisions of this group of animals). Only the cells in which the nucleus and nucleolus were present were considered. Calculation of the total number of magnocellular stained cells was made following the protocol proposed by Rhodes et al. (13) using a correction factor of 0.333 (according to the formula of Abercrombie (1)). In previous studies (16) we have demonstrated that there are no important differences in the nuclear size of the VP-reacting parvicellular neurons when compared to magnocellular ones. We therefore used the same correction factor for both cellular types. Due to the fact that there is no exact limit between the different subdivisions of the pVN, we measured the central part of each.
Calculation of VP-cell surface areas as well as the total number of cells was carried out with an Apple digital planimeter connected to a RCA video system. The values of the parameters obtained were compared statistically using Student’s t _test. Values of p < 0.05 were considered significant. Drawings of the distribution of VP-reacting neurons were performed by means of a camera 1uci‘d a equipped Leitz Laborlux microscope with t h e aid of a low power planar lens. The exact location of the immunoreactive neurons was determined by means of Nissl and cresyl violet stainings or by phase-contrast microscopy.
Morphometry
Results
A) Cellular area. The cellular area was calculated
For the standardization of results the subdivision proposed by Swanson and Kuypers (23) was
by analyzing one hundred randomly selected cells
MORPHOMETRIC CHANGES OF SPECIFIC LOCATED VASOPRESSIN-REACTING PARVICELLULAR IN THE PARAVENTRICULAR NUCLEUS OF THE RAT AFTER ADRENALECTOMY
Table 1
PVN
129
NEURONS
Magnocellular Subdivisions
Group
Females
Males
Females
Males
Cellular Area Commissural k+C ADX+C
N N+C ADX+C
+ Z
+ Z
258.02 259.24 f+ 66.32 59.57 260.00 + 52.68
Posterior
261.29 256.93 +f 60.87 60.67 258.78 + 63.41
I I
Total Number Cells Commissural 39 + 16 35+ 15 I 46 + 19 45f18 I 482 18 44+16 I
215.09 272.82 ++ 62.58 61.82 279.07 + 65.50
275.97 274.51 + +- 10.96 45.67 274.06 +- 48.18
Posterior 1151 + 258 1268 f 221 1270 f 216
1123 + 243 1299 + 238 1281 f 245
Values of Cellular Area and total number of cells obtained in the commissural and posterior magnocellular subdivisions of the paraventricular nucleus. N: normal animals; N + C: normal colchicine treated animals; ADX + C: bilaterally adrenalectomized colchicine-treated animals.
employed. Only the anterior and medial magnocellular subdivisions were grouped together in one subdivision denominated commissural, following Peterson (11) (in view of the scarcity of VP-immunoreactive neurons). Topogruphical study. In normal animals, most VP-reactive neurons were seen in the posterior magnocellular subdivision, the majority densely grouped in its lateral zone (Fig. 1). Occasionally, but not in all the slices, isolated VP-immunoreactive neurons were observed in the parvicellular component, especially in the periventricular subdivision. In the normal colchicine-treated animals the distribution observed in the magnocellular component was similar and showed an increase in reaction intensity. As in the latter group, some VP-immunoreactive neurons in the parvicellular component was observed and these also showed an increase in reaction intensity. In the bilaterally adrenalectomized colchicinetreated animals the characteristics observed in the magnocellular component were similar to those in the above group (Fig. 2). However, there were abundant VP-immunoreactive neurons in the parvicellular component, located exclusively in the periventricular, anterior and medial subdivisions (Figs 3-6). These neurons exhibited clear prolongations, losing the typical rounded shape shown by the normal colchicinetreated animals (Figs 5 and 6). In the periventricu-
lar subdivision they were located primarily in the anterior zone, decreasing progressively as the sections studied became more posterior, to such an extent that in those in which the posterior magnocellular subdivision appeared, only some VP-parvicellular neurons were visualized (Scheme Fig. 2). Morphometric
study. In all the three groups of animals studied, no significant statistical differences were observed between the female and the male animals in any of the subdivisions studied (Tables 1 and 2). In normal animals the values of the parameters obtained in both magnocellular subdivisions were similar, except for a slightly higher cellular area (CA) and a higher number of cells in the posterior magnocellular subdivision (Table 1). In the normal colchicine-treated animals the values in the magnocellular subdivisions were similar to those found in the former group, with no significant differences (Table 1). After bilateral adrenalectomy and colchicine treatment all the parameters analyzed maintained similar values in both magnocelluar subdivisions (Table 1). By contrast, in the periventricular, anterior and medial parvicellular subdivisions, a dramatic increase in the CA and the number of immunoreactive cells was observed with respect to the normal colchicine-treated animals, the differences being statistically significant (p < 0.05) (Table 2). However, in the dorsal and lateral
130
NEUROPEPTIDES
131
MORPHOMETRIC CHANGES OF SPECIFIC LOCATED VASOPRESSIN-REACTING PARVICELLULAR NEURONS IN THE PARAVENTRICULAR NUCLEUS OF THE RAT AFTER ADRENALECTOMY
Fig. 1 Normal animals. Male animal. Note the VP-reactive neurons densely grouped in the posterior magnocellular subdivision (PM). In the medial (MP) and periventricular (PV) parvicellular subdivisions only some isolated VP-reacting neurons can be seen. 320 x (V: Third ventricle. DP: Dorsal parvicellular subdivision). Figs 2 to 6. Bilaterally adrenalectomized animals pretreated with colchicine. Fig. 2 Panoramic view of posterior magnocellular subdivision. As in the normal animals we can see the neurons located in the posterior magnocellular subdivision. Additionally VP-reactive neurons located in the periventricular and medial parvicellular subdivisions can be seen. Only one or two scattered reactive cells can be seen in the dorsal parvicellular subdivision (DP) Female animal. 320 x.
Fig. 3 Panoramic view of anterior (AP) and periventricular 5 VP-immunoreactive parvicellular periventricular neurons. Male animal. 640 x Fig. 5. Female animal. 1280 X). Fig. 6 prolongations (arrow-heads). Medial parvicellular subdivision.
(PV) parvicellular subdivisions. Male animal. 320 x Figs 4 and Some of them show prolongations (Fig. 5. Arrow-heads) (Fig. 4. Several VP-immunoreactive parvicellular neurons showing small Male animal. 1280 X.
parvicellular subdivisions the values of the parameters obtained did not undergo important modifications and were similar to those found in the normal colchicine-treated animals (p > 0.05) (Table 2).
There has been no absolute agreement, however, on the exact location of this neuronal population, some authors having attributed it to the ‘central zone of PVN’ (26), others to the medial parvicellular subdivision (12, 19) and even others to the anterior and medial parvicellular subdivisions (5). On the other hand, it is clear that these parvicellular subdivisions project to the external zone of the median eminence (6, 7, 24, 28) and correspond to the subdivisions where CRF is mainly located (5, 12, 20, 26, 27). The results of this study, together with others obtained previously by us (16), confirm morphometrically the participation of the VP-immunoreactive parvicellular neurons of the anterior and
Discussion In the last decade several papers have shown that adrenalectomy stimulates VP secretion and that VP plays a role in ACTH secretion (3,4,25). The appearance in the PVN of a large population of VP-producing parvicellular cells after bilateral adrenalectomy has also been clearly confirmed (5, 12, 14, 19).
Table 2
PVN Parvicellular
Subdivisions Males
Group
Females
Males
Cellular Area
Females
Number of cells
N+C ADX+C
3 +
148.54 + 26.99 214.58 + 37.55*
Periventricular 144.88 + 23.72 I 211.48 f 39.70* I
N+C
3
ADX + C
3
14254 ?I 31.29 233.77 + 51.82’
Anterior 143.55 f 20.72 230.97 ?I 42.79’
I I
823 37 f 12*
9+3 40 + 15*
N+C ADX+C
+ 3
150.65 + 26.95 236.32 + 53.24’
Medial 154.27 f 26.42 236.67 + 36.67*
I I
38 f 10 67 f 15”
41 ?I 09 65 _+ 12’
ADX+C
194.97 f 43.57 197.73 k 41.27
Dorsal 188.44 k 49.61 189.49 f 46.52
I I
I2 + 04 15 + 04
14 f 03 17 -+ 05
N+C ADX+C
204.51 + 41.74 204.52 + 44.38
Lateral 198.62 t 48.86 207.42 + 41.29
I I
14 + 05 15 + 03
16 + 05 19 +- 05
N+C
22 + 09 80 f 20*
20 k 08 85 + 21*
Parvieellular subdivisions of the paraventricular nucleus. Values of Cellular Area and number of cells. N: Normal animals; N + C: Normal colchicine treated animals; ADX + C: Bilaterally adrenalectomized animals. *p < 0.05 with respect to the N + C animals.
132
Scheme
NEUROPEPTIDES
Drawings of frontal sections from the rostra1 (A) to caudal (H) plane (C to F have been exclusively focussed on the PVN). The drawings show the distribution of the VP-reactive neurons following bilateral adrenalectomy. AC: Anterior commissure. FX: Fornix. OT: Optic tract. pSON: Prechiasmatic Supraoptic nucleus. rSON: Retrochiasmatic Supraoptic nucleus. V: Third ventricle. 1. Commissural magnocellular subdivision. 2. Periventricular parvicellular subdivision. 3. Anterior parvicellular subdivision. 4. Medial parvicellular subdivision. 5. Dorsal parvicellular subdivision. 6. Posterior magnocellular subdivision. 7. Lateral parvicellular subdivision.
MORPHOMETRIC CHANGES OF SPECIFIC LOCATED VASOPRESSIN-REACTING PARVICELLULAR IN THE PARAVENTRICULAR NUCLEUS OF THE RAT AFI-ER ADRENALECTOMY
medial subdivisions as well as in the periventricular parvicellular subdivision in the hypothalamic control of the ACTH secretion. However there now exists morphometric changes either in the lateral and dorsal parvicellular subdivisions or in the two magnocellular subdivisions. These morphological-morphometic results can be easily explained, since it is well known that in studies carried out with retrograde tracers (6, 7, 28) the PVN subdivisions projecting to the EZME are exclusively the anterior, medial and periventricular parvicellular subdivisions. Although traditionally parvicellular neurons have been considered as being clearly smaller than magnocellular neurons, the differences are, perhaps, not as clear especially in some subdivisions (dorsal and lateral parvicellular subdivisions when compared to commissural magnocellular subdivisions). Whilst there are not many publications which offer information about cell size in the PVN, we should emphasize the similarity of our results to those obtained by Sawchenko and Swanson (18), who found even smaller differences between the subdivisions already commented on. The Tables show selective morphometric alterations in the anterior, medial and periventricular and parvicellular subdivisions, consisting in a dramatic increase in the CA and number of cells with respect to the normal colchicine-treated animals. We believe that this indicates a state of cellular activation in response to a potent stimulus such as adrenalectomy. These morphometric results have physiological relevance since statistically significant changes were only seen in those subdivisidns of the PVN which have been implicated in the pituitary control of ACTH secretion, with no changes being observed in either the magnocellular component or in the dorsal or lateral parvicellular subdivisions, regions which do not project to the EZME.
Acknowledgements The authors would like to express their gratitude to Miss E. L. Shorten and to N. Skinner for kindly revising the English version of this manuscript. This work was supported by a DGICYT project, PB86-0213 (Direccibn General de Investigaci6n Cientifica y TCcnica).
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B
NEURONS
133
References 1. Abercrombie, M. (1946). Estimation of nuclear population from microtome sections. Anat. Record 94: 23%247. 2. Antoni, F. A. (1986). Hypothalamic control of adrenocorticotropin secretion: Advances since the discovery of 41-residue corticotropin-releasing factor. Endocr. Rev. 7: 351-378. 3. Antoni, F. A., Palkovits, M., Makara, G. B., Linton, E. A., Lowry, P. J. and Kiss, T. Z. (1983). Immunoreactive corticotropin-releasing hormone in the hypothalamoinfundibular tract. Neuroendocrinology 36: 415-423. 4. Holmes, M. C., Antoni, F. A., Catt, K. J. and Aguilera, G. (1986). Predominant release of vasopressin vs. corticotropin-releasing factor from the isolated median eminence after adrenalectomy. Neuroendocrinolgy 43: 245-251. 5. Kiss, J. Z., Mezey. E. and Skirboll, L. (1984). Corticotropin-releasing factor-immunoreactive neurons of the paraventricular nucleus become vasopressin positive after adrenalectomy. Proc. Natl. Acad. Sci. 81: 1854-1858. 6. Lechan, R. M., Nestler, J. L., Jacobson, S. and Reichlin, S. (1980). The hypothalamic tuberoinfundibular system of the rat as demonstrated by horseradish peroxidase (HRP) microiontophoresis. Brain Res. 195: 13-27. 7. Lechan, R. M., Nestler, J. L. and Jacobson, S. (1982). The tuberoinfundibular system of the rat as demonstrated by immunohistochemical localization of retrogradely transported wheat germ agglutinin (WGA) from the median eminence. Brain Res. 245: 1-15. 8 Makara, G. B., Antoni, F. A., Stark, E. and Kbrteszi, M. (1984). Hypothalamic organization of corticotropin releasing factor (CRF) producing structures. In: Neuroendocrine Perspectives, Vol. 3. Miiller, E. E., McLeod, R. M., eds. pp. 71-119 (Elsevier, Amsterdam). 9 Mezey. E. and Kiss, J. Z. (1985). Vasoactive intestinal peptide-containing neurons in the paraventricular nucleus may participate in regulating prolactin secretion. Proc. Natl. Acad. Sci. USA. 82: 245-247. 10 Negro-Vilar, A., SBnchez-France, F., Kwiatkowski, M. and Samson, W. K. (1979). Failure to detect radioimmunoassayable arginine vasotocin in mammalian pineals. Brain Res. Bull. 4: 789-792. 11. Peterson, R. P. (1966). Magnocellular neurosecretory centers in the rat hypothalamus. J. Comp. Neural. 128: 181-190. 12. Piekut, D. T. and Joseph, S. A. (1986). Co-existence of CRF and vasopressin immunoreactivity in parvocellular paraventricular neurons of rat hypothalamus. Peptides 7: 891-898. 13. Rhodes, C.H., Morrel, J. I. and Pfaff, D. W. (1981). Immunohistochemical analysis of magnocellular elements in rat hypothalamus: Distribution and numbers of cells containing neurophysin, oxytocin and vasopressin. J. Comp. Neurol. 198: 45-64. 14. SBnchez, F., Carretero, J., Rubio, M., Blanco, E., Riesco, J. M. and Vgzquez, R. (1988). Topographical distribution of vasopressin-producing neurons in the paraventricular nucleus of the rat hypothalamus following
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Neuropeptides (1990) 17,127-134 0 Longman Group UK Ltd 1990
Morphometric Changes of Specific Located Vasopressin-Reacting Parvicellular Neurons Paraventricular Nucleus of the Rat After Adrenalectomy F. SANCHEZ*, J. CARRETERO”, F. SANCHEZ-FRANCOt, J. A. JUANES” and R. VAZQUEZ”
J. M. RIESCO’,
in the
E. BLANCO”,
*Department of Human Anatomy and Histology. Avda Campo Charro s/n. Faculty of Medicine. University of Salamanca, 37007 Salamanca, Spain. tEndocrinology Service of the Ramdn y Cajal Hospital, Madrid. (Reprint request to FSl
Abstract-The morphological-morphometric consequences of bilateral adrenalectomy on vasopressin-reacting neurons of the paraventricular nucleus of the rat hypothalamus were analyzed. Bilateral adrenalectomy led to a dramatic increase in the cellular area as well as the number of immunoreactive ceils (when compared to those obtained in normal colchicinetreated animals) in the neurons located in the anterior, medial and periventricular parvicellular subdivisions of the paraventricular nucleus. By contrast, no changes were observed in either the dorsal or lateral parvicellular subdivisions or in any of the magnocellular subdivisions of the paraventricular nucleus.
Introduction In recent years various peptides have been implicated in the hypothalamic control of corticotropic hormone (ACTH) secretion; the ACTH releasing factor (CRF) and vasopressin (VP) have received particular attention (4, 8, 25, 27), although other peptides, such as VIP (9) are gradually being implicated in this process. Different papers, including studies from our laboratory, have highlighted the important role of Date received 9 March 1990 Date accepted 28 May 1990
VP-producing neurons in the parvicellular regions of the hypothalamic paraventricular nucleus (PVN) in the secretory control of pituitary ACTH release (5, 12, 14, 19, 26). These parvicellular neurons do not normally express VP immunoreactivity (20). Morphometry has been shown to be an adequate technique to valorate the consequence of different experimental states on the central nervous system. Accordingly, in this study the poseffects of bilateral sible morphometric adrenalectomy on VP-producing neurons of the PVN were evaluated in an immunocytochemicalmorphological-morphometric study. 127
128 Material and Methods
The animals were housed in standard laboratory conditions: seasonal light-dark cycle, temperature 22 + 2°C R.H. 50 + 5%, and food and water ad libitum. Thirty male and female Sprague-Dawley rats were used, divided into three groups: 1) Normal animals (n = 10,5 per sex); 2) Animals pretreated with 90 t~,gof colchicine dissolved in 15 ~1 of saline and administered in the lateral ventricle by stereotaxic surgery 24h prior to sacrifice (n = 10, 5 per sex); 3) Bilaterally adrenalectomized animals (15 days) under ketamine anaesthesia (n = 10, 5 per sex). Like those of group 2, the rats were pretreated with colchicine (24h prior to sacrifice). Following adrenalectomy (dorsal approach), the animals’ drinking water was supplemented with sodium chloride (0.9%) and sucrose (5%). The completeness of adrenalectomy was verifed by necropsy. After sacrifice by decapitation, the hypothalamic-hypophyseal block was fixed by immersion in Bouin-Hollande solution and embedded in paraffin. Serial 5 pm frontal sections were obtained with a microtome. fmmunocytochemzktry
After inhibiting endogenous peroxidase by Passing the samples through a methanol bath Hz02 (22), the sections were pretreated with normal Swine serum (DAKO). Following this, the PAP immunocytochemical method was applied (21). As primary serum, anti-VP Serum was employed (10, 17) at a dilution of l/1000. The rest of the sera (anti-rabbit immunoglobulin swine serum and PAP soluble complex) were supplied by DAK0 (see 14). The reaction was visualized using a 0.05% solution of 3-3’ diaminobenzizidine in TRIS-HCL buffer containing 0.01% hydrogen peroxide. Preabsortion test with VP (SIGMA) and substitution test of the primary serum by normal rabbit serum were carried out. After preabsortion or substitution test the immunoreaction was completely abolished.
NEUROPEPTIDES
(never more than three per subdivision and section) in both the parvicellular and magnocellular subdivisions (100 cells in each subdivision, per sex and different experimental group of animals were analyzed, with the exception of the parvicellular subdivisions of group 1 in which, with the technique employed, the VP-reactive neurons were very scarce and presented a very weak reaction intensity). B) Number of reacting cells. The number of cells was calculated by analyzing each subdivision, sex and group of animals, with the exception of the parvicellular subdivisions of group 1 (given the scarcity and the weakness of immunoreaction of the VP-reacting neurons in the parvicellular subdivisions of this group of animals). Only the cells in which the nucleus and nucleolus were present were considered. Calculation of the total number of magnocellular stained cells was made following the protocol proposed by Rhodes et al. (13) using a correction factor of 0.333 (according to the formula of Abercrombie (1)). In previous studies (16) we have demonstrated that there are no important differences in the nuclear size of the VP-reacting parvicellular neurons when compared to magnocellular ones. We therefore used the same correction factor for both cellular types. Due to the fact that there is no exact limit between the different subdivisions of the pVN, we measured the central part of each.
Calculation of VP-cell surface areas as well as the total number of cells was carried out with an Apple digital planimeter connected to a RCA video system. The values of the parameters obtained were compared statistically using Student’s t _test. Values of p < 0.05 were considered significant. Drawings of the distribution of VP-reacting neurons were performed by means of a camera 1uci‘d a equipped Leitz Laborlux microscope with t h e aid of a low power planar lens. The exact location of the immunoreactive neurons was determined by means of Nissl and cresyl violet stainings or by phase-contrast microscopy.
Morphometry
Results
A) Cellular area. The cellular area was calculated
For the standardization of results the subdivision proposed by Swanson and Kuypers (23) was
by analyzing one hundred randomly selected cells
MORPHOMETRIC CHANGES OF SPECIFIC LOCATED VASOPRESSIN-REACTING PARVICELLULAR IN THE PARAVENTRICULAR NUCLEUS OF THE RAT AFTER ADRENALECTOMY
Table 1
PVN
129
NEURONS
Magnocellular Subdivisions
Group
Females
Males
Females
Males
Cellular Area Commissural k+C ADX+C
N N+C ADX+C
+ Z
+ Z
258.02 259.24 f+ 66.32 59.57 260.00 + 52.68
Posterior
261.29 256.93 +f 60.87 60.67 258.78 + 63.41
I I
Total Number Cells Commissural 39 + 16 35+ 15 I 46 + 19 45f18 I 482 18 44+16 I
215.09 272.82 ++ 62.58 61.82 279.07 + 65.50
275.97 274.51 + +- 10.96 45.67 274.06 +- 48.18
Posterior 1151 + 258 1268 f 221 1270 f 216
1123 + 243 1299 + 238 1281 f 245
Values of Cellular Area and total number of cells obtained in the commissural and posterior magnocellular subdivisions of the paraventricular nucleus. N: normal animals; N + C: normal colchicine treated animals; ADX + C: bilaterally adrenalectomized colchicine-treated animals.
employed. Only the anterior and medial magnocellular subdivisions were grouped together in one subdivision denominated commissural, following Peterson (11) (in view of the scarcity of VP-immunoreactive neurons). Topogruphical study. In normal animals, most VP-reactive neurons were seen in the posterior magnocellular subdivision, the majority densely grouped in its lateral zone (Fig. 1). Occasionally, but not in all the slices, isolated VP-immunoreactive neurons were observed in the parvicellular component, especially in the periventricular subdivision. In the normal colchicine-treated animals the distribution observed in the magnocellular component was similar and showed an increase in reaction intensity. As in the latter group, some VP-immunoreactive neurons in the parvicellular component was observed and these also showed an increase in reaction intensity. In the bilaterally adrenalectomized colchicinetreated animals the characteristics observed in the magnocellular component were similar to those in the above group (Fig. 2). However, there were abundant VP-immunoreactive neurons in the parvicellular component, located exclusively in the periventricular, anterior and medial subdivisions (Figs 3-6). These neurons exhibited clear prolongations, losing the typical rounded shape shown by the normal colchicinetreated animals (Figs 5 and 6). In the periventricu-
lar subdivision they were located primarily in the anterior zone, decreasing progressively as the sections studied became more posterior, to such an extent that in those in which the posterior magnocellular subdivision appeared, only some VP-parvicellular neurons were visualized (Scheme Fig. 2). Morphometric
study. In all the three groups of animals studied, no significant statistical differences were observed between the female and the male animals in any of the subdivisions studied (Tables 1 and 2). In normal animals the values of the parameters obtained in both magnocellular subdivisions were similar, except for a slightly higher cellular area (CA) and a higher number of cells in the posterior magnocellular subdivision (Table 1). In the normal colchicine-treated animals the values in the magnocellular subdivisions were similar to those found in the former group, with no significant differences (Table 1). After bilateral adrenalectomy and colchicine treatment all the parameters analyzed maintained similar values in both magnocelluar subdivisions (Table 1). By contrast, in the periventricular, anterior and medial parvicellular subdivisions, a dramatic increase in the CA and the number of immunoreactive cells was observed with respect to the normal colchicine-treated animals, the differences being statistically significant (p < 0.05) (Table 2). However, in the dorsal and lateral
130
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MORPHOMETRIC CHANGES OF SPECIFIC LOCATED VASOPRESSIN-REACTING PARVICELLULAR NEURONS IN THE PARAVENTRICULAR NUCLEUS OF THE RAT AFTER ADRENALECTOMY
Fig. 1 Normal animals. Male animal. Note the VP-reactive neurons densely grouped in the posterior magnocellular subdivision (PM). In the medial (MP) and periventricular (PV) parvicellular subdivisions only some isolated VP-reacting neurons can be seen. 320 x (V: Third ventricle. DP: Dorsal parvicellular subdivision). Figs 2 to 6. Bilaterally adrenalectomized animals pretreated with colchicine. Fig. 2 Panoramic view of posterior magnocellular subdivision. As in the normal animals we can see the neurons located in the posterior magnocellular subdivision. Additionally VP-reactive neurons located in the periventricular and medial parvicellular subdivisions can be seen. Only one or two scattered reactive cells can be seen in the dorsal parvicellular subdivision (DP) Female animal. 320 x.
Fig. 3 Panoramic view of anterior (AP) and periventricular 5 VP-immunoreactive parvicellular periventricular neurons. Male animal. 640 x Fig. 5. Female animal. 1280 X). Fig. 6 prolongations (arrow-heads). Medial parvicellular subdivision.
(PV) parvicellular subdivisions. Male animal. 320 x Figs 4 and Some of them show prolongations (Fig. 5. Arrow-heads) (Fig. 4. Several VP-immunoreactive parvicellular neurons showing small Male animal. 1280 X.
parvicellular subdivisions the values of the parameters obtained did not undergo important modifications and were similar to those found in the normal colchicine-treated animals (p > 0.05) (Table 2).
There has been no absolute agreement, however, on the exact location of this neuronal population, some authors having attributed it to the ‘central zone of PVN’ (26), others to the medial parvicellular subdivision (12, 19) and even others to the anterior and medial parvicellular subdivisions (5). On the other hand, it is clear that these parvicellular subdivisions project to the external zone of the median eminence (6, 7, 24, 28) and correspond to the subdivisions where CRF is mainly located (5, 12, 20, 26, 27). The results of this study, together with others obtained previously by us (16), confirm morphometrically the participation of the VP-immunoreactive parvicellular neurons of the anterior and
Discussion In the last decade several papers have shown that adrenalectomy stimulates VP secretion and that VP plays a role in ACTH secretion (3,4,25). The appearance in the PVN of a large population of VP-producing parvicellular cells after bilateral adrenalectomy has also been clearly confirmed (5, 12, 14, 19).
Table 2
PVN Parvicellular
Subdivisions Males
Group
Females
Males
Cellular Area
Females
Number of cells
N+C ADX+C
3 +
148.54 + 26.99 214.58 + 37.55*
Periventricular 144.88 + 23.72 I 211.48 f 39.70* I
N+C
3
ADX + C
3
14254 ?I 31.29 233.77 + 51.82’
Anterior 143.55 f 20.72 230.97 ?I 42.79’
I I
823 37 f 12*
9+3 40 + 15*
N+C ADX+C
+ 3
150.65 + 26.95 236.32 + 53.24’
Medial 154.27 f 26.42 236.67 + 36.67*
I I
38 f 10 67 f 15”
41 ?I 09 65 _+ 12’
ADX+C
194.97 f 43.57 197.73 k 41.27
Dorsal 188.44 k 49.61 189.49 f 46.52
I I
I2 + 04 15 + 04
14 f 03 17 -+ 05
N+C ADX+C
204.51 + 41.74 204.52 + 44.38
Lateral 198.62 t 48.86 207.42 + 41.29
I I
14 + 05 15 + 03
16 + 05 19 +- 05
N+C
22 + 09 80 f 20*
20 k 08 85 + 21*
Parvieellular subdivisions of the paraventricular nucleus. Values of Cellular Area and number of cells. N: Normal animals; N + C: Normal colchicine treated animals; ADX + C: Bilaterally adrenalectomized animals. *p < 0.05 with respect to the N + C animals.
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Scheme
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Drawings of frontal sections from the rostra1 (A) to caudal (H) plane (C to F have been exclusively focussed on the PVN). The drawings show the distribution of the VP-reactive neurons following bilateral adrenalectomy. AC: Anterior commissure. FX: Fornix. OT: Optic tract. pSON: Prechiasmatic Supraoptic nucleus. rSON: Retrochiasmatic Supraoptic nucleus. V: Third ventricle. 1. Commissural magnocellular subdivision. 2. Periventricular parvicellular subdivision. 3. Anterior parvicellular subdivision. 4. Medial parvicellular subdivision. 5. Dorsal parvicellular subdivision. 6. Posterior magnocellular subdivision. 7. Lateral parvicellular subdivision.
MORPHOMETRIC CHANGES OF SPECIFIC LOCATED VASOPRESSIN-REACTING PARVICELLULAR IN THE PARAVENTRICULAR NUCLEUS OF THE RAT AFI-ER ADRENALECTOMY
medial subdivisions as well as in the periventricular parvicellular subdivision in the hypothalamic control of the ACTH secretion. However there now exists morphometric changes either in the lateral and dorsal parvicellular subdivisions or in the two magnocellular subdivisions. These morphological-morphometic results can be easily explained, since it is well known that in studies carried out with retrograde tracers (6, 7, 28) the PVN subdivisions projecting to the EZME are exclusively the anterior, medial and periventricular parvicellular subdivisions. Although traditionally parvicellular neurons have been considered as being clearly smaller than magnocellular neurons, the differences are, perhaps, not as clear especially in some subdivisions (dorsal and lateral parvicellular subdivisions when compared to commissural magnocellular subdivisions). Whilst there are not many publications which offer information about cell size in the PVN, we should emphasize the similarity of our results to those obtained by Sawchenko and Swanson (18), who found even smaller differences between the subdivisions already commented on. The Tables show selective morphometric alterations in the anterior, medial and periventricular and parvicellular subdivisions, consisting in a dramatic increase in the CA and number of cells with respect to the normal colchicine-treated animals. We believe that this indicates a state of cellular activation in response to a potent stimulus such as adrenalectomy. These morphometric results have physiological relevance since statistically significant changes were only seen in those subdivisidns of the PVN which have been implicated in the pituitary control of ACTH secretion, with no changes being observed in either the magnocellular component or in the dorsal or lateral parvicellular subdivisions, regions which do not project to the EZME.
Acknowledgements The authors would like to express their gratitude to Miss E. L. Shorten and to N. Skinner for kindly revising the English version of this manuscript. This work was supported by a DGICYT project, PB86-0213 (Direccibn General de Investigaci6n Cientifica y TCcnica).
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