Vasopressin in reaggregated cell cultures of the developing hypothalamus

Brain Research Bulkrin, Vol.

12,pp. 307-313,1984.0

0361-9230/&t $3.00 + .OO

Ankho InternationalInc. Printedin the U.S.A.

Vasopressin in Reaggregated Cell Cultures of the Developing Hypothalamus’ M. F. D. NOTTER,2 Departments

of Anatomy

D. M. GASH, C. D. SLADEK and Neurology,

Received

University

14 November

AND S. L. SCHAROUN

of Rochester,

Rochester,

NY 14642

1983

NOTTER, M. F. D., D. M. GASH, C. D. SLADEK AND S. L. SCHAROUN. Vasopressin in reaggregated cell culrures of the developing hyporhalamus. BRAIN RES BULL 12(3) 307-313, 1984.-A microsystem for rotation-mediated aggregate cell culture studies has been devised to examine vasopressin (VP) biosynthesis of developing rat hypothalamus. Trypsindispersed hypothalamic tissue was placed into 24 well tissue culture dishes and VP content of culture medium and cells was measured over time by a radioimmunoassay. Reaggregates formed within 4 hr when rotated at 70 rpm in a humid CO* incubator. Nineteen days post coitus (dpc) hypothalamic reaggregates had 336 pg VP/loB cells while the medium showed 260 pg VP/ml after four days. Measurable VP was seen in fetal tissue after ten days while comparable amounts of VP were present in one day neonatal hypothalamus over this same period. Morphological examination of reaggregates indicated the presence of viable cells throughout the cell mass after ten days of culture. Co-cultivation studies with dispersed posterior pituitary indicated that reaggregates from one day neonate hypothalamus had significantly increased VP levels when co-cultured with one day neonatal posterior pituitary; however, this effect was not seen with 19 dpc co-cultures. These data demonstrate that development of neurosecretory activity of discrete regions of the hypothalamus can be examined early in vitro in a reaggregate cell culture system. Vasopressin

Reaggregate

Cell culture

Posterior pituitary

vaginal smear used to determine day zero of pregnancy. Birth normally occurred in the morning or early afternoon on the 21 day post-coitus (dpc) and this was considered postnatal day zero. Hypothalamic tissue and pituitaries were microdissected from 12 to 19 dpc fetuses and one day postnatal (dpn) pups following procedures described elsewhere [5,6]. Essentially, using the developing optic tract, neurohypophysial stalk and the Circle of Willis as landmarks, the entire anterior hypothalamus was removed. Tissue was placed in sterile, cold, calcium-free, magnesium-free buffer (CMF: 0.15 M NaCl, 0.008 M N&HPOI, 0.0027 M KCl, 0.0015 M KHPOI, 0.026 M HaHCO,, with 0.1% glucose, 100 j&ml streptomycin, 2.5 pg/ml fungizone). In later experiments, if tissue was to be kept for more than one hour before processing, it was placed in sterile beakers with bicarbonate-buffered Eagle’s minimal essential medium aerated with CO,/O, mixture to maintain a proper pH. The tissue was cut into small (l
PRIMARY cell culture of nervous tissue has proved to be a useful tool in developmental neurobiological studies. In par-

ticular, since the external environment of the cells can be controlled, the roles of epigenetic factors such as nerve growth factors, hormones, and neurohumors can be assessed readily while the effect of co-cultivation with various cell types and the interactions between them can be evaluated. Recently, Honegger and co-workers [9, 10, 11,151 have used a multidisciplinary approach to studies of development of rat brain in vitro. Using a rotation-mediated aggregating cell culture system, they showed that reaggregated fetal brain cells undergo cellular differentiation representative of central nervous system development in vim; that reaggregates synthesize and release neurotransmitters in culture and that this system is useful for pharmacological and toxicological studies in developmental neurobiology. In this report we describe a micro-system for rotationmediated aggregation cell culture experiments and present preliminary data of vasopressin detection by aggregating cultures of fetal and neonatal rat hypothalamus maintained alone or in co-culture with posterior pituitary cells. METHOD

Tissue Preparation All animals used came from our breeding colonies of Long-Evans and Wistar-Lewis rats. Animals were allowed to breed from 1800-0800 hr with the detection of sperm in the

‘Supported by USPHS Grant NS15109 (DMG). 2Requests for reprints should be addressed to Dr. M. F. D. Notter, Department of Anatomy, Box 603, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642.

307

30X

NOT’I’ER. GASH. SLADEK

pipet to obtain dispersed cells. Cell number and viability were calculated on an aliquot of the cell suspension using 0.1% erythrocin dye exclusion and a hemocytometer counting chamber. The procedure routinely provided I x IO” cells for each anterior hypothalamus dissected from a 19 dpc fetus with cell viability greater than 85%. Trypsinization of one day old hypothalamic tissue yielded the same range of cells while treatment of early embryonic tissue such as 12 dpc and I5 dpc hypothalamus required less trypsin (0.1% for five minutes). Trypsinization of the whole pituitary as well as the posterior pituitary routinely yielded cell dispersions with 95% or greater cell viability.

Radioimmunoassay for vasopressin quantification was performed as previously described [6]. Briefly, cells and media from each tissue culture well were centrifuged at 1000 rpm in an IEC centrifuge for five minutes and the supernatant stored frozen at -20°C for direct assay. Pelleted cells were homogenized in one milliliter of a 0.25% acetic acid solution and then centrifuged. All samples were analyzed for vasopressin using an antiserum developed by van Wimersma Greidanus [21] with a sensitivity of two pglassay tube. Rotation Mediatrd Cdl Culturr

Cell reaggregation procedures outlined by Moscona [16] and used by Honegger and Richelson [8,9] were modified for use in a microsystem. Dispersed hypothalamic cells were seeded at a concentration of IX 106/ml into individual siliconized wells of multiwelled tissue culture plates (Costar 16 mm diameter per well; 24 wells per plate) in one milliliter of growth medium (described below). Posterior pituitary cells were seeded at lo” cells per well unless otherwise specified while complete pituitaries were plated at 2.5X lo” cells per well. In some cases, when the number of hypothalamic cells was increased twofold, the number of pituitary cells was likewise increased twofold in the same experiment to keep the cell to cell ratio the same. When seeded together. volumes were adjusted to 1.5 ml per well. Growth medium contained Eagle’s minimal essential medium, supplemented with 10% fetal calf serum, 0.6% glucose, 0.1% glutamine, 100 &ml streptomycin and 2.5 pg&l fungiwne and was changed evecv four davs. Plates were olaced on a Junior orbit shaker (Labline) and’rotated at 70 rpm in a humid (5% CO,, 95% air) incubator. Aggregates could be viewed and photographed quite easily with an inverted microscope while still in culture in individual wells of a culture plate. Manipulation of aggregates could be done on a single well basis within the same plate.

Tissue was processed for histological examination by fixation within individual wells of the culture dish. Reaggregates were fixed in Zamboni’s futative, embedded in paraffin, sectioned at 6 pm and stained with cresyl violet acetate. Reaggregates embedded in Epon araldite were fixed in Karnovsky aldehyde fixative [ 121 sectioned at I pm and stained with methylene blue. Stdsticul

unalysis

The data were analyzed by a non-parametric sign test [ 131 in which the values of VP content obtained when tissue was co-cultured were compared to values obtained by adding the VP content from tissues cultured separately.

TABLE

A\ci) SC’HAR0I.i’.

I

CELL NUMBER PER BRAIN FOR DISSECTED RA’I I-ISSIJES DIFFERENT STAGES OF DEVELOPMEN I --.--

I:! day Embryonic I3 day Embryonic 15 day Embryonic 17 day Fetal 19 day Fetal I day Neonatal 7 day Neonatal 19 day Fetal I day Neonatal I day Neonatal 3 day Neonatal Adult Neonatal

A’l

Ventral diencephalon Ventral diencephalon Ventral diencephalon Anterior hypothalamus Anterior hypothalamus Anterior hypothalamus Anterior hypothalamus Whole Pituitary Anterior Pituitary Posterior Pituitary Posterior Pituitary Posterior Pituitary

*Average t- standard error of the mean.

RESULTS

Trypsinization of hypothalamic as well as pituitary tissue yielded a single cell suspension of primarily spherical cells. Cell size in one day neonatal hypothalamus ranged between IO-18 pm. Cell yield and viability were optimal if tissue was processed immediately after dissection and when the concentration and duration of trypsin was carefully controlled. As seen in Table 1, cell numbers per block of tissue increased with age of the donor.

Hypothalamic cells as well as pituitary cells formed aggregates within four hours when maintained in rotation culture. At 24 hours, spheroids of hypothalamic cells were about 150 pm in diameter as measured through the culture plate with an ocular micrometer inserted in the eyepiece of an inverted tissue culture microscope (Fig. 1). Comparably sized reaggregates were formed by 12 dpc through one dpn fetal tissue; however hypothalamic cells from 5 dpn animals did not form typical reaggregates and cellular viability was lower than that seen for younger cells. Reaggregates of pituitary tissue tended to be smaller since they were seeded into culture wells at a lower concentration (Fig. 2). Plating more than 2x 10” hypothalamic cells/l6 mm well yielded reaggregates that were larger than 150 pm after 24 hours, a finding which agrees with Moscona’s early reports l16f on concentration-dependent reaggregation of cells from various parts of the embryo.

Histological examination of I9 dpc reaggregates maintained in culture for four days revealed a well organized cellular mass within and throughout the reaggregate (Fig. 3a, b) when stained for nissl substance. Twelve dpc hypothalamic reaggregates in culture for ten days showed good cellular organization and a similar morphology even after this extended time in vitro (Fig. 4). No pyknosis was seen indicating viable, healthy cells. When a number of reaggregates

REAGGREGATED CELL CULTURES

FIG. 1. Hypothalamic ceU reaggregatesfroma i9 day fetus maintainedfor ten days in culture. Cells {I x 16) were rotated at 70 rpm in vitro and spheroidsroughly 150pm in diameter formed within four hours. Rsmgregates were photographed directly through _the tissue culture dish. 250x.

wcm r&ryp&ized

after four days in culture, zi celf f25untand

test for viability indicated

that 9lS of the cefls in the reaggregates were viahle while cell number was sliitly increased over the initial level of cells seeded into the culture wells.

Reaggregates of 12 dpc hy~~~ic tissue produced high levels of vasopressin in the media after ten days of culture: (822 pg/ml) while hypothalamic cells from 19 dpc fetuses contained vasopressin through eight days in vitro (Table 2). VP levels in tissue after four days were 336.5 prJloS cells while 507.7 pg VPIloB cells could be measured &er eight days. VP was ako detected in tissue culture media after these same times (Table 2). Since media was changed after four days in vitro, the hig&er levels of VP in reaggregates after eight days may indicate VP synthesis and/or storage. Aggregates of hypothalamic cells from one day neonates were also examined for VP and as seen in Table 2, VP levels in the tissue were comparable to that seen for fetal hypothalamus over eight days in cutture. interestingly the media contained signifiiautly higher levels of VP during this same period, possibly reflecting an increased synthesis and release. Pituitary tissue from 19 dpc fetuses showed high levels of VP in culture for four days while posterior pituitq reaggregates from one day neonates assayed for VP hormone content after four and ten days contained large amounts of VP possibly present in degenerating nerve terminals or pituicytes (Table 3). Media collected from all pituitary cultures also had representative levels of VP. Effect of Co-Cultivation of Pituitary Cd& With Hyp~t~#~~rni~ Reaggregates ~y~th~~us

was co-cubred

with

dispersed pituitary

FIG. 2. One day neonatalposterior pituitary res@regatts four days in culture. Cells(1x 106)formed spheroids 100pm in diameterunder the conditions described for Pip. 1. 238x. tissue and formed mixed reaggregates in cuJture. When one day neonatal hypothalamus was cultured with one day posterior pituitary, there was a significant increase in VP content of co-cultured reaggregates after four days in vitro (Table 4). This increase was significantly greater than the sum of the VP values attained from hypo&&mus and posterior pituitary cultured ~de~nden~y @<0X%2;Table 2 and 3).

NOTTER,GASH,SLADEK

ANI)

SC'HAROk~h

FIG. 3. a. Reaggtegate of one day neonatal hypothalamic cells in culture for four days. The spheroid shows as a well formed cell mass. Parathn section, 600x. b. One day neonatal reaggregate of hypothalamic cells after four days of culture. Paraffin section, 1200.

FIG.4. Reaggregate of 12 day fetal hypothalamic cells in culture for ten days. Nuclei of cells exhibit dispersed chromatin with prominent dense nucleoli (arrow). No pyknosis is evident. Cells are from the center of the reaggregate. One pm thick, plastic embedded section. 4500x.

311

REAGGREGATEDCELLCULTURES

TABLE

2

HYPOTHALAMUS IN REAGGREGATE CELL CULTURE

19 dpc (1 x l@ 19 dpc (1 x 10 1 day neonate 1 day neonate

pg VP/cells*

Days in Vitro

Age cells) ceils) (1 x l@ cells) (1 x l@ cells)

4

pg VP/ml medium

336.5 ? 39.7 507.7 2 285.3

8 4

103.7 f 201.7 f

10

20.2 132.4

*Average it standard error of the mean of several experiments, point per experiment.

TABLE

260.3 2 119.5 *

11.4 13.5

2242.7 ? 436.1 1246.9 2 286.2

2-4 samples per time

3

PITUITARY TISSUE IN REAGGREGATE CELL CULTURE

pg VP/cells*

pg VP/ml medium

4 4

495.7 2 4.3 601.8 f 203.7

131.0 f 7.0 316.9 + 112.1

10

330.3 k 218.3

215.3 -r- 119.8

Days in Vitro 19 dpc whole pituitary (200,000 cells) 1 day neonate, posterior pituitary 030,000 cells) 1 day neonate, posterior pituitary 030,000 cells)

*Average -C standard error of the mean of several experiments, ment.

TABLE

2-4 samples per time point per experi-

4

CO-CULTURES OF REAGGREGATED HYPOTHALAMUS WITH PITUITARY TISSUE

Tissue

Cell Number

Days In Vitro

pg VP/cells*

19 dpc hypothalamus + 19 dpc pituitary

1 x 1W cells 2x 105cells

4

1 dpn hypothalamus + 1 dpn posterior pituitary 1 dpn hypothalamus + 1 dpn posterior pituitary

1X 1tP cells 8x l(r cells

4

2199.3 + 756.6t

1x l@ cells 8x 10’ cells

10

657.7 ? 516.2

1x IOBcells 1x 105cells

4

138.6 k

1 X 108 cells 1 X 105cells

10

90.1 f

19 dpc hypothalamus + 1 dpn posterior pituitary 19 dpc hypothalamus + 1 dpn posterior pituitary

396.5 *

73.9

pg VP/ml medium

131.0 ?

7.0

1811.0 -t 435.8

1641.0 f

90.0

24.0

122.1 f

32.0

16.0

730.0 k 282.8

*Average k standard error of the mean for several experiments. 2-4 samples per time point per experiment. Wgnificantly different (0.02
312

NOTTER,

VP concentration in the media from co-cultures was somewhat lower after four day in vitro in some experiments although there was no difference seen in other studies. After ten days in culture, the VP content of co-reaggregates was still high. This phenomenon of increased VP levels from cocultures of neonatal hypothalamus and posterior pituitary was not seen when I9 dpc hypothalamus and 19 dpc pituitary were co-cultured (Table 4) nor when neonatal hypothalamus was co-cultured with neonatal anterior pituitary (data not shown). In an attempt to establish a critical time in the development of fetal tissue, 19 dpc hypothalamus was cocultivated with one day neonatal posterior pituitary. As seen in Table 4, the neonatal neurohypophysis had no effect on VP content of I9 dpc hypothalamus. DISCUSSION

The mammalian hypothalamic-neurohypophyseal system is a well-defined neuroendocrine system which serves as an excellent model for investigating neural development. Previous studies [6,18] by our group have focused on the normal in vile development of magnocellular vasopressin neurons of the rat supraoptic nucleus. These studies indicated that VP concentrations in the hypothalamic neurosecretory system increase 3-6 fold daily from 21 pglanimal on fetal day 17 to 5984 pg/animal at birth. In the present study we examined the in vitro development of vasopressin neurons of fetal and neonatal rat anterior hypothalamus by determining the amount of vasopressin present in these neurons when maintained in a cell reaggregate culture system. Twelve day fetal through one day neonatal hypothalamic tissue form reaggregates rapidly and these reaggregates secrete vasopressin throughout ten days in culture. Histologically these reaggregates contained viable cells throughout the cell mass over this time period. The level of VP released into the media from I9 dpc hypothalamic reaggregates ( I x I Oficells per one hypothalamic unit) was comp;irable to that measured by Sladek and Knigge [17] from one adult rat hypothalamohypophyseal system over nine days in explant culture (350 pg VP/24 hr). Denzeau et ul. [2] could detect VP at a somewhat lower level in culture medium from 1x 10” dispersed 19 dpc rat hypothalamic cells after three days i/l vitro. HOW-

GASH, SL,ADEK AND SCHAROUN

ever, they were unable to detect positive immunohistochemical staining for VP in these cells during this time. Our results were somewhat different in that we were able to detect :I considerable amount of VP within hypothalamic cells b) RIA by culturing the same number of IY dpc hypothalamic cells in a reaggregate culture. Interestingly, VP content of hypothalamic tissue could be enhanced by co-cultivation of one day neonatal hypothalamus with one day neonatal posterior pituitary. This was a significant finding not reproduced when anterior pituitary was substituted for posterior pituitary nor when I9 dpc hypothalamus was co-cultured with one day neonatal posterior pituitary cells. These observations may indicate a critical time in vasopressin neuron development in which these cells may be stimulated by the posterior pituitary. Indeed there i, evidence that target tissues in the central and peripheral nervous systems produce factors that influence the maintenance and survival of neurons which interact specifically with them [I]. In this study, cultures of the posterior lobe indicated the presence of vasopressin in cells and media over the duration of culture. These findings appear to reflect the situation observed in ,lil,o. Experiments involving the destruction of the hypophyseal stalk in the rat demonstrated that the discharge of vasopressin from the degenerating neural lobe ofthe pituitary continued over at least a five day period [3,14] and pituicytes and endothelial cells were shown to have phagocytic and excretory activity during this time 131. In a series of studies with fetal rat brain reaggregates maintained in culture for a long period, Honegger and COworkers were readily able to follow the effects of dibutyrl cyclic adenosine monophosphate and ascorbic acid on the activity of neurotransmitter metabolizing enzymes [9]. neurotransmitter biosynthesis, storage and release [IO], myelination [ 1.51and thyroid hormone action in developing brain [I I]. With the present study. we have shown that the reaggregate culture system can be adapted to a microassay of neurosecretory activity of selected regions of fetal hypothalamus and that this microsystem can be employed quite readily to examine the effects of co-cultivation with target tissues on brain development at critical time\

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