Thyroid hormones reversibly suppress somatostatin secretion and immunoreactivity in cultured neocortical cells

Brain Revearch, 328 (1985) 259-270 Elsevier

259

BRE 10565

Thyroid Hormones Reversibly Suppress Somatostatin Secretion and Immunoreactivity in Cultured Neocortical Cells R()BERT A. PETERFREUND 3. PAUL E. SAWCHENK()-" and WYLIE VALE I I Clayton Foundation Laboratories fbr Peptide Biology, "Developmental Neurobiology Laboratoo', The Salk Institute, 10010 North Torr O' Pines Road, La Jolla, ('A 92037 and ~Department o fNeuroscience~, Univers'ity <)["C'alilbrnia, San Diego, La Jolla, ('A ~20~)3 (U.S.A.) (Acceptcd June 5th, 1984) Key words: somatostatin --- cerebral cortex c e l l s - primary c u l t u r e - thyroid h o r m o n e s immunohistochemistry - - radioimmunoassay

Thyroid hormone effects on brain somatostatin-like immunoreactivity (SRIF-LI) were studied in an in vitro model system. Serum was removed from the nutrient culture medium of fetal day-18 rat cerebral cortex cells maintained in primary, long-term, dispersed monolayer culture. Chronic administration of either T) or T 4 in serum-free medium was associated with suppressed release of SRIF-LI into the culturc medium (36-43 h accumulation), cell content of peptide and acute release in response to potassium-induced depolarization. Suppression was dose-dependent with an IC~(, of less than 1 nM for F:~. The most dramatic effects were obscrved for K +-induced release. Thirty-five to 50% suppression was typically observed with T~ at a near maximum dose (3 nM). Reverse T~ and diiodotyrosine were less potent and effective than T 3. TRIAC and diiodothyronine also possessed significant suppressive activity. 'I"3 suppression of release depended on duration of pretreatment. Administered for less than 16 h, T~ failed to significantly suppress K+-in duccd release, but significant suppression was observed for pretreatment periods of 16 h or longer, Indirect fluorescent immunohistochemical examination revealed a reduction in the number of cells positively stained tk)r SRIF-LI in Y~-treated dishes relative to controis. Upon removal of T 3 and subsequent recovery in serum supplemented medium for 24 h, F¢-treatcd and control cells exhibited similar levels of SRIF-LI release and cell content. T~-treated and control cells incorporated [3H] Icucine into trichloracetic acid precipitable counts to similar extents. Dcxamethasonc and several sex steroids failed to modify the effects of T~ and did not independently inlluence SRIF-LI levels. Acute cycloheximide administration did not reverse 'l'~ effects. The data indicate that primary brain cell cultures may be useful models to examine direct peripheral hormone actions on nervous tissue. Thyroid hormones suppress SRIF-LI levels in a dose, time and structure-dependent manner, which appears to be reversible. The findings are consistent ssith a possible integration of peripheral hormone and brain peptide physiology. INTRODUCTION

P r e p a r a t i o n s of C N S tissue m v i t r o p r o v i d e m o d e l s to e x a m i n e t h e d i r e c t a c t i o n of t h y r o i d h o r m o n e s at

T h y r o i d h o r m o n e s h a v e well r e c o g n i z e d i n f l u e n c e s

t h e level of t h e n e r w m s s y s t e m . M o d u l a t i o n of n e u -

o n b o t h t h e d e v e l o p i n g a n d t h e m a t u r e brainLL Per-

ronal d i f f e r e n t i a t i o n , m y e l i n f o r m a t i o n a n d glyco-

t u r b a t i o n of t h y r o i d s t a t u s h a s b e e n a s s o c i a t e d with

p r o t e i n s y n t h e s i s , as f u n c t i o n s o f t h y r o i d h o r m o n e

altered

brain

morphology,

eters,

cognitive

functions

neurochemical and

param-

levels, h a v e b e e n d e s c r i b e d in in v i t r o systems2,3,1(,.~2.

pat-

W e h a v e p r e v i o u s l y d e s c r i b e d a p r i m a r y c u l t u r e sys-

b i n d i n g sites for

t e m of fetal rat b r a i n e s t a b l i s h e d to e x a m i n e t h e pro-

t h y r o i d h o r m o n e s in c e n t r a l n e r v o u s s y s t e m ( C N S )

d u c t i o n a n d s e c r e t i o n o f C N S p e p t i d e h o r m o n e s :7.

ternsl,S, Jl Jg.> 25.31,33.34 A l t h o u g h

behavior

tissue h a v e b e e n identified'~,el,22,3s,-~% s e v e r a l investi-

C u l t u r e d cells f r o m fetal day-18 rat c e r e b r a l c o r t e x

gators have suggested

nervous system

m a i n t a i n e d for e x t e n d e d p e r i o d s in vitro s e c r e t e t h e

changes observed following alterations of thyroid

p e p t i d e s o m a t o s t a t i n in r e a d i l y m e a s u r a b l e q u a n t i -

status in vivo c o u l d b e s e c o n d a r y to c h a n g e s in pe-

ties as d e t e r m i n e d by r a d i o i m m u n o a s s a y ( R I A ) . In

r i p h e r a l p a r a m e t e r s , such as c i r c u l a t i n g h o r m o n e o r n u t r i e n t levels 14,20,37.

the p r e s e n t series o f e x p e r i m e n t s , t h e a p p a r e n t long-

that some

t e r m viability a n d r e s p o n s i v e n e s s of t h e s e c u l t u r e s

Corre~'pondence: W. Vale, Peptide Biology Laboratory, The Salk Institute. P. (), Box 8580(I, San l)iego, ('A 9213.',, t J.S. A O006-,S993:S5:$03.3i) @ 1985 Elsevier Science Publishers B.V. (Biomedical Division)

260 have been exploited to examine the effects of chronic thyroid hormone treatment on a candidate physiologic parameter of certain brain cells, the content and release of somatostatin. MATERIALS AND METHODS

Reagents Reverse T 3 (3',5',3-triiodothyronine), cycloheximide and D-actinomycin were purchased from Calbiochem (La JoUa, CA). fl-estradiol, testosterone, progesterone, dexamethasone and all other thyronine analogues were purchased from Sigma Chemical Co. (St. Louis, MO). Propranoloi-HCl (Inderal) was from Ayerst (New York, NY). Pentex crystalline bovine serum albumin (cBSA) was obtained from Miles Laboratories (Elkhart, IN). [3H]leucine was purchased from New England Nuclear (Boston, MA). Synthetic peptides were provided by Dr. J. E. Rivier.

Buffers and media H E P E S - K r e b s - R i n g e r - b i c a r b o n a t e - g l u c o s e solution containing 0.1% cBSA and 30#g/ml bacitracin (Calbiochem) ( H K R B G ) , and HEPES dissociation buffer (HDB) were prepared as previously described 27. High potassium H K R B G (59 mM) had KCI isotonically substituted for NaCI. Serum supplemented culture medium, SSM, consisted of H E P E S buffered Dulbecco's Modified Eagle medium (HDME) supplemented with 10% (v/v) fetal calf serum (Kansas City Biologicals, Lenexa, Kansas), 10% (v/v) horse serum (Grand Island Biological Co., Grand Island, NY), and antibiotics. D-glucose (1-1.5 g/liter) and L-glutamine (200-300 mg/liter) were added to the base medium. Serum free culture medium (SFM) consisted of H D M E which was supplemented with 0.1% (w/v) cBSA, 6 g/liter additional glucose, 1.2 g/liter additional glutamine and antibiotics.

Cell culture Preparation of primary long-term dispersed cell cultures of fetal rat cerebral cortex was as previously described 2v. Briefly, fetal Sprague-Dawley rats were removed from their etherized mothers via an abdominal incision on the 18th day of gestation. From each fetus a thin slice of rostral, lateral cerebral

cortex was removed from both hemispheres. Tissue was enzymatically dispersed with collagenase 11 (Worthington Biochemicals Corp.. Freehold. N J) and DNase II (Sigma). Cells were washed, counted and plated in 1.5 ml serum-supplemented medium on poly-D-lysine-coated tissue culture dishes at a densit~ of 5 × 106 cells/35 mm dish On the 3rd and 5th or 6th day in culture, cells were fed with serum-supplemented medium diluted to 5% (v/v) horse serum and 5c~ (v/vl fetal calf serum with HDME.

Pretreatment of cells On the 7th day in culture, cells were washed twice with 1 ml each of SFM and then incubated for 28-32 h in 1.5 ml SFM. Medium was then replaced with 1.5 ml fresh SFM per dish and cells received test substances or diluent only (control) for 37-43 h (pretreatment period). Treatment groups consisted of 4-6 dishes in most experiments. Thyronine compounds were initially dissolved in 0.1 N N a O H at 1-2 mg/ml prior to dilution with SFM. All drugs and hormones were sterile filtered (Millex-GV. Miltipore, Bedford, MA) prior to addition to the dishes as 30fold concentrates in a volume of 50 ul.

Secretion expertments For experiments examining stimulated secretion. t=0 is defined as the time of administration of 59 mM K + . Dishes of cells were washed 3 times with 1 ml of H K R B G . and subsequently allowed to equilibrate for 1 h (t = -1 to t = 0 h) in 0.75 ml H K R B G in an incubator at 37 °C, 100K humidity and 7.5% CO,. With the exception of time course experiments, drugs or hormones were routinely readded to the appropriate dishes at the start of the equilibration period (t = -1 h) to reduce the possibility of escape from pretreatment effects. After equilibration, cells were treated with 59 mM K + in H K R B G for 1 h (t = 0 to t = + 1 h). Following stimulation, dishes of cells were either extracted for peptide content or were allowed to recover in serum-supplemented medium, As a measure of unstimulated secretion during a 1 h period in H K R B G (5.9 mM K + ). the release of SRIF-Li by control dishes during the equilibration period prior to stimulation was routinely assessed. This level of release closely approximates non-stimulated release in cells previously allowed to equilibrate and is

261

hereafter referred to as basal release.

Sample preparation SFM samples (1.5 ml) from the treatment period were collected with a glass pipette into glass tubes on ice containing 50 ul 0.5 M EDTA, heated in a water bath for 5 rain at >85 °C and frozen until assayed. HKRBG samples were collected into an equal volume of 2 N HOAc in glass tubes on ice, heated and then dried in a Speed Vac (Savant Instruments, Hicksville, NY). For assay, samples were resuspended in RIA buffer containing phenol red. and neutralized. Cell content samples were collected after stimulation. Following removal of incubation medium, 0.75 ml cold 2 N HOAc was added to each of the dishes which were then rapidly frozen on dry ice. After thawing, cellular material was scraped from the dishes and collected into glass tubes. A rinse of 0.75 ml 2 N HOAc/dish was pooled with the first collection. Samples were heated, sonicated for 10 s and the tubes were centrifuged at 2000 g. The pellet was discarded and the supernatant dried and saved for RIA. (?ell content samples were initially resuspended in 0.1 ml 1(I mM HOAc prior to dilution with R1A buffer.

[ ~H/Leucine incorporation Minimum essential medium with a reduced leucine content (Leu- HMEM) was prepared with a Select Amine kit (Gibco). The base medium was supplemented with 10 mM HEPES, 2 g/liter additional glucose and 240 rag/liter additional glutamine. Leucine content was approximately 2% of concentration in complete medium. Following pretreatment with or without T 3, cells were washed twice with Leu HMEM containing 0.1% cBSA and then incubated in 0.75 ml fresh medium containing 25/aCi/ml [3H] leucine for 3 h. Cells were washed twice and cellular material was removed from dishes with two washes of a solution of 1% Nonidet P 40 (NP40) (Sigma) in 77 mM NaCI. 0.25 ml 50% trichloracetic acid (TCA) was added io the pooled washes (final concentration = lit% TCA) and samples were heated at greater than 85 °C for 90 s in an H:O bath. Samples were cooled, vortexed and l()(t ul aliquots were filtered in duplicate through 0.45 um filters (Millipore, HAWP). Filters were washed with 4 × I ml 10c~'r

TCA; dried and precipitated counts were determined in 11 ml Aquasol-2, (NEN) with a Beckman LS-8000 scintillation counter (Beckman Instruments, Fullerton, CA). Quenching was monitored with an external gamma source (the H #) and found to be essentially uniform for all samples. Background count precipitation was determined by adding [3H]leucine to solubilized material from an unlabeled dish of cells followed by precipitation with TCA. Cycloheximide (20/ag/ml) was added to some dishes concurrent with [3H]leucine. T~ was readded to appropriate dishes at the start of the labeling period.

lmmunohistochemical studies Histochemical studies of SRIF-I,I in vitro were done in parallel with secretion experiments described above. After removal of serum from the media, 6 dishes were treated with T3 (3 nM, 43 h) and 6 served as controls. At the end of the pretreatment period, the dishes were rinsed twice for 3-4 min in HDB and then fixed for 15 rain in freshly prepared 4% (w/v) paraformaldehyde lind 0.(15o~, (vol/vol) glutaraldehyde in 0.1 M borate buffer at pH 9.5. Fixed dishes were then rinsed in two changes (ll) min each) phosphate-buffered saline (PBS) and incubated for 48 h at 4 °C in a 1:2000 dilution of rabbit anti-SRIF serum (Immunonuclear Corp., Stillwater. MN) in PBS that also contained 0 . 3 ~ Triton X-101) and 2~'/~ normal goat serum. After again rinsing in two changes of PBS, the primary antiserum was localized by exposing the cells for 45 min at room temperature to a 1:200 dilution of affinity purified, fluorescein isothiocyanate-conjugated goat anti-rabbit lgG (Tago, Inc., Burlingame, CA). After two final rinses in PBS the cells were coverslipped with a buffered glycerol mountant 15, lind analyzed using epi-illumination fluorescence microscopy. The number of SRIF-stained cells contained in each culture dish was estimated by counting the number of cells that fell within a square (0.16 ram/side) counting grid at 250 × magnification as the dish was moved through two complete orthogonal traverses of the 22 x 22 mm square coverslip. Fhe obtained values were multiplied by the inverse of the fraction of the area of the dish that was covered by the traverses. Counts were made without knowledge of treatment status. After the SRIF-stained cells were counted, the total number of cells per dish was esti-

262

200

mated by first staining the cells with ethidium bromide. which is a fluorescent label for Nissl sub-

A

160

1~/~"~

stance 3°. then averaging the n u m b e r of cells counted in each of 10 counting grid placements that were

59mM K +

evenly spaced along two orthogonal traverses of the coverstip and. finally, multiplying bv the reciprocal of the fraction of the area of the dish s u b t e n d e d by

co)

(t)

120

one counting grid. Preliminary experiments in which these counting procedures were repeated on individual dishes yielded highly reproducible esumates.

0

E _J

As controls for antiserum specificity, additional

80

dishes of untreated cells were prepared for immunohistochemistry using anti-SRIF sera that were prein-

orU)

cubated for 16-24 h with l.O mg/ml synthetic SRIF40

14 or SRIF-2g.

~./~

BASAL ........................................

RIA and statisucs r-,~/~,

0

I

0.01

~

~ TIT,,,

I

~

i

irliH

0.1

I

I

r

i

,IT,I,

RI A data and statistical analyses were carried out

l

10

[T31 nM

usmg programs on the Salk Institute V A X computer as previously described zT. Between group, comparisons for secretion data were performed with the D u n -

360

can test for multiple comparisons. Within group variance was always homogeneous. Data is reported as mean secretion or content _ S.E for the dishes in a treatment group. Comparison of groups in i m m u n o -

320

280

-

240

-

histochemical studies was carried out with an F tesL RESULTS

200

-

Effects of iodothyronines: dose, time and structure dependence

160 -

In initial experiments. 3 parameters were folt,r"

120 80 40

0

0.01

0.1 [T 31 nM

I

I0

Fig. 1. A, B: response of cortex cells to doses of T 3. Cells were prepared as described in Materials and Methods and then pretreated for 40 h with addition of medium only or one of several doses of T3. A: response to 59 mM K* during a 1 h incubation (e~--O): Basal secretion (. . . . ) was 22.5 _+ 1 fmol/dish in untreated ceils. B: 2 N acetic acid extraction of cells for contents of SRIF,LI ( A - - A ) and SFM culture medium content after 39 h (I----B) IC50 for inhibition of SRIF-LI levels was less than 1 nM T3 for al 3 parameters. Significant suppression of

lowed: SRIF-LI content of SFM collected at the end of the 36-42 h p r e t r e a t m e n t period, cellular eontem of SRIF-LI, and response to a depolarizing stimulus, 59 mM K +. after pretreatment, Subsequently, ontv the response to 59 m M K + was assessed Chronic exposure to T 3 in SFM was associated with a dose-dependent ~ p p r e s s l o n of all 3 parameters (Fig 1), with the most dramatic effects observed for the response to 59 m M K + in H K R B G . In a typical experiment, significant suppression of potassiumstimulated release occurred bv (J.3 nM T 3 with an IC m of less than 1 nM. Chronic administration of the .

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.

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.

.

.

S R I F - L I ( P < O.O1 or P < 0.051 w a s o b s e r v e d for [3 at I nM

{culture medium h 0.3 nM (cell contents) and 0.3 nM (response to 59 mM K~ ~.

.

.

.

.

.

.

.

263 possessed

240

significant

suppressive

activity

(Table

I A , B ) . Interestingly, t r e a t m e n t with high doses of D-T4 also was associated with suppression of S R I F - L ! 200

secretion (not shown).

bTuorescence

g { g '6

160

59mM K+

staining

and

immmtohistochemical

atlaly,sis R a d i o i m m u n o a s s a y findings w e r e e x t e n d e d using i m m u n o h i s t o c h e m i c a l m e t h o d s to slain for S R I F - L I

120 -

-5

in vitro. In both u n t r e a t e d and T~-treated dishes,

d.

round to o v o i d cells c o n t a i n i n g p u n c t a t e , fluorescent

rr" o3

granules were seen e v e n l y dispersed t h r o u g h o u t the

80-

dishes (Fig. 5). Cells w e r e ll)-14,urn in d i a m e t e r and typically gave rise to 2 - 4 40-

branch

BASAL

•

................................................

extensively.

processes that did not

Varicose

S R I F - p o s i t i v e fibers

were also p r e s e n t , and could occasionally be seen to e m e r g e from stained cell bodies. P r e a d s o r p t i o n of 0 -

r--

o

'

i

lO

i 20

3

0[

410

-q 50

280

H O U R S OF P R E T R E A T M E N T WITH T 3 (3 nM)

Fig. 2. Time course of the response to T 3 (3 nM). A stock solution of T 3 was prepared and stored in frozen, sterile aliquots. 1~ was added at the indicated points prior to stimulation with 59 mM K ~ . In this experiment T~ was not added at the start of the equilibration period. Response to 59 mM K + ( 0 - - 0 ) is depicted. Basal secretion in untreated dishes was 28.5 ± 1 fmol. dish ( . . . . ). Significant suppression (P < 0.01) was observed only fi)r ~1"~pretreatment periods of 16 h or hmgcr. Significant suppression of culture medium content was observed only for the longest pretreatment period (42 h) (not shown). Cell content was not determined in this experiment.

240 T4

200 c-

._m "E3

160 O

-n just m a x i m a l c o n c e n t r a t i o n of T) (3 nM) typically suppressed K ~ - i n d u c e d secretion to 5 0 - 6 0 % of control levels.

120-

59raM K+

IX: 03

80

T h e d u r a t i o n of T 3 p r e t r e a t m e n t also i n f l u e n c e d the o b s e r v e d effects (Fig. 2). S h o r t - t e r m t r e a t m e n t

40

BASAL

of I or 6 h was not associated with d i m i n i s h e d response to 59 m M K +. Significant suppressive effects were o b s e r v e d for p r e t r e a t m e n t periods lasting 16 or m o r e hours with m a x i m u m suppression at 42 h, the longest p r e t r e a t m e n t p e r i o d tested. T h y r o x i n e (L-T4) m i m i c k e d the effects of T ; with similar efficacy and p o t e n c y (Fig. 3).

R e v e r s e 1"~

(rT~) also suppressed S R I F - L I s e c r e t e d in r e s p o n s e to 59 m M K* (Fig. 4) and S R I F - L l l e v e l s in the m e d i um and cell extracts (not shown). H o w e v e r , rT 3 was a p p a r e n t l y less p o t e n t and effective than T> D i i o d o L-tyrosine was inactive (Table 1C), but t r i i o d o t h v roacetie acid ( T R I A C )

and 3 , 5 - d i i o d o - l , - t h v r o n i n e

0

I- ~

0

'

I

' I

0 1

1 [Thyronine]

~

'

~

lO nM

T

~

7

100

Fig. 3. Response to doses of 1~ and "l-4. ('ells w e r e p r e t r e a t c d with medium only (control) or o n e of several doses of T 3 or T 4 for 38 h. Response to 59 mM K" is depicted (I~--tD "I~, T4). Significant suppression of response was observed byT~ = 0.1 n M (P < 0.011 and ]-4 ~ l.l) n M (I' < 0.01). Basal

secretion was 33.3 ± 1 fmol/dish ( . . . . ). In the same experiment (not shown), culture medium content (37 h accumulation) was significantly' suppressed by, T~ = 1.0 nM ( 1' < 0.01 ) and also byT 4 = 1.0 nM ( P < 0.05). For cell conten/, significant suppression was obsereed by 1"~ = 0.1 nM (P < ). )¢,) and T 4 = 1(1 nM ( P < 0.01).

264

280

tion m response to 59 mM K + were measured in control and T3-treated dishes (Table l l t A ) . After snmulation, dishes were returned to SSM for -~24 hours Irecovery period), stimulated again and then cell

240

peptide was extracted. Following an initial, significant suppression of SRIF-LI release. F , - l r e a t e d cells appeared to recover content of p e p n d e as well as the

200 t-

ability to secrete at or near control levels. In fact, the second response to 59 mM K*. after recovery m SSM. greatly exceeded that elicited by the first stimulation. In the second protocol, extractable content of

160 O

E -5 i

It

\

59mM

K-'-

SRIF-LI in cells not permitted to recover tTable

120 -

IIIB. line 2), and in cells allowed to recover for 24 h

r'r O9

in SSM lTable IIIB. line 4), were compared. After recovery, T~-treated cells contained near control lev-

80-

els of peptide and this level exceeded the quantity of extractable S RIF-LI in cells not permitted to recov4O

0

I BASAL F- ~

0

er. As a measure of the general viability of the cells i

03

~ [Thyronine]

10 nM

"

i

I00

Fig. 4. Response to doses of "I"3and rT3. Cells were pretreated with culture medium only I control) or one of several doses of T 3 or rT3for 38.5 h, Response to 59 mM K+ is depicted (0----0 q?3, (D---O rT3). Significant suppression was observed by T~ = 0.3 nM (P < 0.051 and rT.~= 30 nM (P < 0.01 ). Basal secrenon was 18.6 _+ fmol/dish ( - - - L In the same experiment (not shown), culture medium content (37.5 h accumulation)was significantly suppressed by T3 = 1.0 nM (P < 0.011. No suppression was observed with rT3. Cell content was significantly suppressed by T~ = 1.0 nM (P < 0.05) and rT~ = 100 nM (P < 0.05/.

anti-SRlF serum with an excess of either synthetic SRIF-14 or SRIF-28 completely blocked the staining of cells and fibers in u n t r e a t e d dishes. Significantly fewer SRIF-positive cells were estimated in T~-treated (3 nM, 43 h) dishes, relative to controls (Table II). Though not quantified, treated dishes also appeared to contain substantially fewer stained fibers. Neither the total cell n u m b e r (TableII) nor the general appearance of cells in the culture dishes was significantly altered by T 3 treatment.

Reversibility of the effects of T3 and cell viability Recovery experiments using two protocols were performed to assess possible reversibility of thyroid h o r m o n e effects on SRIF-LI (Table III). In one protocol, culture m e d i u m content of SRIF-LI and secre-

under the SFM experimental procedures, we examined protein synthesis m control and T,-treated cells. Cells pretreated with "1-3 for 43 h incorporated [3H]leucine into T C A precipitable protein to the same extent as untreated cells, although cycloheximide added during the labeling incubation almost completely prevented label incorporation (Table 1V).

Combined effects q[" 7~ and other treatments Several agents were tested either alone or in combination with T 3 for their effects on SRIF-LI content and/or secretion in cortex cells [Table VI. The peripheral hormones testosterone, estradiot, progesterone and the glucocorticoid dexamethasone did nol markedly modify SRIF-L1 levels when administered alone for extended periods. When administered concurrently with T3, these agents failed to either amplify or reverse the effect of a near maximal dose of the thyronme. Propranolol, a /~-adrenergic antagonist used therapeutically in thyrotoxicosis .~5,36. also failed to reverse the T3-induced suppression of SRIF-LI levels. Administration of cycloheximide (20 #g/ml) during the equilibration period prior to stimulation, and subsequently during stimulation with 59 m M K +. also failed to reverse the effects of T~ pretreatment (Table V I A l . Small but significant effects on SRIFLI secretion were observed for dishes treated with cycloheximide alone. C o n c u r r e n t administration of

265

TABI,E I

Comparison o f the efl~'cts o f T~ and analogues In addition to rT~ and T~, we tested other analogues of T~ for activity in the brain cell culture system. Cells were prctreated with T 3 or analogues for 43 h (A], 42.5 h (B), or 42 h (C). SRIF-LI in 59 mM K + stimulating buffer was measured. Basal secretion in control dishes was 32.4 + 3 (A), 43.8 _+ 1 (B) and 44.8 ± 2 (C). Trealme~'lI

Response to 59 m M K* t)nol/dish + S. E.

A Control 3'~ (3 nM] TRIAC(I nM) I'RIAC ( I0 nM)

269.6 _+ 8 119.5 ± 5* 158.6 ___ 16" 112.1 ±5*

44.3 58.8 41.6

259.3 ± 172.4 ± 138. l ± 2118.9 ± 204.8 ± 148.8 ±

66.5 53.3 811.6 78.9 57.4

B

('ontrol 1 3 (0.3 nM) T 3 (3 nM) 3'.5' diiodoq -thyronine ( 1 nM) 3'.5' diiodo-i -thyronine ( lfl nM) 3',5' diiodo-i-thyroninc (100 riM)

C Control "I~ (3 nM) Diiodotyrosinc (1 nM) Diiodotyrosinc (10 nM) Diiodotyrosmc (111{)nM)

18 2* 9* 16~** 11 *** 5*

318.1 ± 15 18,1.8 ± 6* 289.9 ± 17"* 305.8 _+ 8** 299.5 _+ 6**

% oj'control

58.1 91.1 96.1 94.2

* = different from control (P < 11.1)1): ** = not significantly different from control (P > 0.05): *** = different from control [P < 0.05).

D-actinomycin

T 3 (3 n M ) a d m i n i s t e r e d in s e r u m - s u p p l e m e n t e d

(5 !~g/ml) for t h e e n t i r e p r e t r e a t m e n t p e r i o d (42.5 h)

m e d i u m was less e f f e c t i v e in s u p p r e s s i n g S R I F - L I

was a s s o c i a t e d with a loss o f T 3 - s u p p r e s s i o n c o m -

levels t h a n identical t r e a t m e n t in S F M , R e s p o n s e

T 3 and

either

cycloheximide

or

p a r e d to cells t r e a t e d only with c y c l o h e x i m i d e or

levels to 59 m M K + in c o n t r o l d i s h e s m a i n t a i n e d in

D - a c t i n o m y c i n ( T a b l e V I B ) . H o w e v e r , cells t r e a t e d

S F M w e r e similar to t h o s e f o u n d in dishes c u l t u r e d in

with c y c l o h e x i m i d e or D - a c t i n o m y c i n h a d very low

SSM d u r i n g the r o u t i n e p r e t r e a t m e n t p e r i o d .

levels of s e c r e t i o n c o m p a r e d to c o n t r o l c o n d i t i o n s ,

Cells m a i n t a i n e d in S F M r e t a i n e d p r e v i o u s l y d o c u -

Fig, 5. A: fluorescence photomicrograph of cultured neocortical neurons stained with ethidium bromide, which is a fluorescent marker for Nissl substance, to illustrate the appearance and density of cells in the in vitro model system. Excitation wavelength centered fit 635 nM (Leitz filter system N2). Magnification: × 260. B: fluorescence photomicrograph of a single cultured neocortical neuron stained positively with an antiserum against SRIF. Three processes can be seen to emerge from this neuron: two are thicker and have the appearance of dendrites, while a finer (axon-like) one is varicose and of uniform diameter. An autofluorescent (non-specifically stained) cell may be seen in the lower right quadrant. Excitation wavelength centered at 435 nM (Leitz filter system I:). Magnification: × 66{1.

266 TABLE II lzf]ect of T~ treatment on SR1F-immunofTuorescence in vitro The possibility that treatment with T 3 was associated with a loss ol cells from the dishes was tested by staining the total cell population with ethidium bromide. Using indirect immunofluorescence techniques, the possibility that T 3 would reduce the number of cells stained specifically for somatostatin was also examined. Cells were pretreated for 43 h. In parallel dishes from the same experiment. T 3 suppressed response to 59 mM K + from 262.1 _+ 10 to 140.3 z 5 fmol/dish 154% of control). Results are representative of two other experiments. Treatment

Total no. cells~dish (+ S.E.)

Control

4,493,555 + 191,341

T 3 (3 nM)

4.688,981 + 235,282**

% o f control

SRIF-positive cells~dish (+_S.E.~

c4 of control

778 +_ 127 104

297 z 60*

38

* = significantly different from control P < 0.01 : ** = not significantly different from control P > 0 0~.

m e n t e d r e s p o n s i v e n e s s t o c a r b a c h o l ( c o n t r o l . 24.4 ~-

sion of 3 p a r a m e t e r s , cell c o n t e n t o f p e p t i d e , secre-

1 f m o l / d i s h ; c a r b a c h o l ( 1 0 0 / ~ M ) , 40.0 _+ 2 f m o l / d i s h .

t i o n d u r i n g t h e p r e t r e a t m e n t p e r i o d a n d r e l e a s e in re-

p < 0.01126,29 a n d to t h e p e p t i d e C R F 2s ( c o n t r o l . 30.1

s p o n s e to a d e p o l a r i z i n g s t i m u l u s , is o b s e r v e d at low

+- 2 f m o l / d i s h ; C R F ( 1 0 0 n M ) 44.7 _+ 4 f m o l / d i s h . P <

n a n o m o l a r d o s e s o f a d m i n i s t e r e d T:~ o r Ta. It is likely,

0.01, u s i n g s y n t h e t i c r a t C R F ) .

however, that the effective concentrations of thyroid

DISCUSSION

than the calculated values due to possible binding of

h o r m o n e s a v a i l a b l e to t h e cells a r e s o m e w h a t less hormones to serum albumin added to the culture meO u r e x p e r i m e n t s e x p l o i t t h e l o n g - t e r m viability a n d a p p a r e n t h e a l t h o f d i s p e r s e d b r a i n cells m a i n t a i n e d in p r i m a r y c u l t u r e to d e m o n s t r a t e a n e f f e c t of

d i u m (N. A l e x a n d e r . p e r s o n a l c o m m u n i c a t i o n ) . Duration of treatment

with thyroid hormones

a p p a r e n t l y i m p o r t a n t in d e t e r m i n i n g

is

response be-

peripheral hormones from the thyroid gland on a cen-

c a u s e a d m i n i s t r a t i o n o f T3 f o r 16 h o r less is n o t a s s o -

tral n e r v o u s s y s t e m p e p t i d e , s o m a t o s t a t i n . S u p p r e s -

c i a t e d w i t h s u p p r e s s i o n . T h i s t i m e c o u r s e ~s consis-

TABLE III SRIF-LI secretion and content after removing 7~: recovery from suppression We tested possible reversibility of the effects of T 3, Cells received either vehicle only or T3 pretreatment. A: after stimulation, control and T3-treated cells were allowed to recover for 24 h in SSM, stimulated again and cell peptide was extracted. Results are representative of two other, identical, experiments. B: after the first stimulation, some dishes were extracted. Other dishes were allowed to recover in SSM, stimulated again and extracted. Results are expressed as fmol/dish + S.E~ Stimulus was 59 m M K + i n H K R B G . T 3 concentration was 3 nM in experiment A and 5 nM in experiment B. T 3 was administered for 42 h in both experiments. Basal secretion was (fmol/dish) 28.5 + 1 (A) and 24.4 _+ 1 (B). Treatment

A Culture medium content First stimulation 24 h recovery Second stimulation Cell content after 2nd stimulation Stimulation Cell content ~, (parallel dishes, 24 h recovery) Stimulation Cell content after stimulation

% o f control

Control

T~

222.0 _ 17 216.9 + 17

158.0 +_ 6 103.2 z 7

71.2* 47.6*

340.4 +__27 224.9 +_ 17

293.5 +__15 250.6 +_ 14

86.2" * 111.4 "~

142.2 +_ 6 213.3 ~_ 18

68.4 z 4 158.4 +__12

48.1 ~ 74.3 ~ +

449.6 _~ 18 278 3 - 21

426.2 ~ 20 241.6 *_ 17

94.8 ' ' 86.8 ~=

* = different from control, (P < 0.01); ** = not significantly different from control. (P > 0.05); "*~ = different from control, ( e < 0.05).

267 TABLE IV

Incorporation of/-;H/leucme into TCA precipitable counts,
Treatment

Precipitable counts/rnin

% of control

(ontrol T> (3 nM) Cvcloheximide 120 ug/ml)

351.61",1 ± 12,797 376.415 _+ 9,214

107.9

15.590 ± 573

4.5

tent with a mechanism of action for thyroid hormones initially mediated by nuclear binding and modifications of RNA synthesis -q,2:. Unlike pituitary cells ~s,

brain cells exhibit an apparently stable response to thyroid hormones because short-term treatment with cycloheximide fails to reverse the effect of long-term T 3 treatment. The effects of T3 on brain cell SRIF-L1 can be prevented with long-term blockade of protein or RNA synthesis. However. it is somewhat difficult to interpret these latter data owing to the overall depression of SRIF-L1 levels and to the possibility of toxicity associated with extended exposure to these metabolic poisons. Non-specific cytotoxicity does not appear to be responsible for the observed effects of thyroid hormones on brain cell SRIF-LI because T 3 did not significantly alter the number or the appearance of cultured cells. Moreover, treatment effects are reversed upon removal of T3 and a vigorous recovery is indicated by the elevated levels of peptide measured after 24 h in SSM recovery medium for control cells, as well as for cells originally exposed to T> A generalized suppression of cellular metabolism also seems inadequate to account for T3 effects because treated and untreated cells exhibit similar degrees of labeled

TABLE V

('ombined effects o f T; and other treatments on S R I F - L I content and secretion We tested the effects of agents from several other regulatory systems on SRIF-LI levels. Test substances were administered either alone or in combination with T 3. Treatments were administered for 42.5 h in A, 41 h in B, 40.5 h in C, and 43 h in D. Results are expressed as fmol/dish + S.E. Basal secretion was 39.9 _+ 1 fmol/dish in A, 24.4 + 1 in B, 25.3 ± 1 in C, and 32.3 ± 3 in D.

Treatment

Response to .59 m M K +

Culture medium

Cell extract SRIF-L1 content

A Control T 3 (3 nM) Testosterone ( ltl nM) Testosterone + T~ Propranolol (5/~M) + "1'~

202.6 121.3 191.3 128.8 124.9

± 7 ± 5* _+ 6** ± 3 *.~' _+ 5*-"

427.8 377.0 462.1 354.4 328.4

NT

B

Control ~1"~(5 nM) Dexamethasone (20 nM) J'~ + Dexamethasone

142.2 68.4 157.8 79.6

± ± ± ±

6 4* 8** 2 *:,

NT

C

Control T 3 (3 nM) fl-Estradiol (lt) nM) /:LEstradiol + T~

195.8 122.0 176.6 115.8

± ± ± ±

7 6* 8** 6 *~'

486.3 395.2 469.7 393.6

269.6 119.5 230.8 128.5

_+ 8 _+ 5* ± 5*** ± 5 *,a

D Control "I'~ (3 nM) Progesterone ( 10 nM) "F~ + Progesterone

NT

± 9 ± 12" ± 6*** ± l(I *,~ _+ 10 *.b

__+30 __+ 17"** + 25** _____11"**-,,

213.3 158.4 229.5 147.3

_+ 18 _+ 12"** _+ 6** ± 9 *~,

299.3 264.7 314.3 250.7

__+7 __+ 10"** ± 16"* +__ 13,, *

NT

different from control, (P < 0.01), * = not s~gmficant[v, different from control, (P > ll.05,): *** = different from control, (P < 0.05): ,* = not significantly different from T 3 only (P > 0.05); b = different from T 3 only (P < 0.111): NT = not tested.

268 TABLE VI Effects of cycloheximide and o-actinomycin on T3 suppression of SRIF-LI secretion

Times indicate time of addition before stimulation at t = 0. A. We tested the hypothesis that the effects ofT 3 are labile and depend on ongoing protein synthesis. Cells were treated with T3 or medium only at -43 h and then received one of two doses of cycloheximide during the equilibration period prior to stimulation, (at -1 h), and also during stimulation with 59 mM K + buffer: Basal secretion was 33.6 ± 3 fmol/dish. B. We tested the hypothesis that the effect of T3depends on protein or mRNA synthesis during some portion of the incubation. Cells were pretreated for 42.5 h with combinations of T3, D-actinomycinor cycloheximide as indicated. Basal secretion in control dishes was 32.9 + 2 fmol/dish. Significant suppression (P < 0.01) of secretion compared to Control was observed for all treatments. Dishes of cells treated with o-actinomycin or cycloheximide (20 #g/ml) did not exhibit further suppression when also treated with T3. However, secretion in dishes treated with cycloheximide (0.2 #g/ml) was further significantlysuppressed by T~. Treatment

Response to 59 m M K + fmol/dish ± S.E.

% of control

A Acute cycloheximide treatment

Control T3 (3 nM) (-43 h) Cycloheximide (20/~g/ml)(-1 h) T3 + cycloheximide (20/~g/ml) Cycloheximide (1/tg/ml) (- 1 h ) T3 + cyeloheximide (1/~g/ml) B

236.6 + 19 126.5 ± 9* 178.2 +_6* 117.8 ± 4*.... 196.5 + 18** 103.4 ± 4*,***

53.5 75.3 49.8 83.1 43.7

Long-term T3, cycloheximide and D-actinomycin treatment

Control T3 (3 nM) (-42.5 h) Cycloheximide (20/~g/ml) (-42.5 h) T3 + cycloheximide (20/~g/ml) Cyeloheximide (0.2#g/ml) (-42.5 h) T3 + cycloheximide (0.2ktg/ml) n-actinomycin (5/~g/ml) (-42.5 h) T3 + D-actinomycin(5 #g/ml)

193.3 + 5 t21.8 +_5 39.1 +_2 39.9 +_2 165.0 ± 7 108.6 4_-3 71.7 z I 72.0 ± 3

63.0 20.2 20.6 85.4 56.2 37.1 37.3

* = significantly different from control (P < 0.01); "* = not significantly different from control fP > 0.05L *** = significantly different from corresponding treatment with cycloheximide alone (P < 0.01). amino acid incorporation into T C A precipitable pro-

alone or in combination with T 3. Reproductive

tein. Specificity of label precipitation was demonstrated by nearly complete elimination of count pre-

steroid hormones and the glucocorticoid dexamethasone failed to modify basal or T3-influenced levels o! SRIF-LI at the doses tested. Thus, neocortex soma-

cipitation in extracts of cells treated with the protein synthesis inhibitor cycloheximide. In general, response to thyronine analogues bv peptidergic cortical n e u r o n s appears to follow structure activity relationships reported for other systems ~7. However, in vitro systems may lack clearance mechanisms, binding proteins or other factors present in intact animals which could mask biological activity of thyronine derivatives. It is also possible that small degrees of impurity, racemization, differences in brain versus peripheral T 3 receptor selectivity or non-specific effects could account for the suppression of SRIF-LI levels associated with high doses of some analogues. The p r o n o u n c e d effects of thyroid h o r m o n e s on somatostatin levels assayed in vitro encouraged an evaluation of response to h o r m o n e s or h o r m o n e analogues from other major peripheral systems, either

tostatin levels in vttro do not appear to be influenced in a general way by peripheral hormones. The data also suggest that fl-adrenergic receptors are not revolved in this action of T,. The present findings appear to have several implications. First. dispersed fetal rat brain ceils in longterm primary culture may provide a potentially useful model system to examine and specify chronic influences on CNS function. The viability and apparent functional integrity exhibited by brain cells following extended m a i n t e n a n c e in relatively well defined culture media would tend to facilitate experimentation. Although large quantities of peptide do not appear to be stored by these cells, as determined by immunocytochemistry or cell extraction, readily measurable quantities are released in various conditions. Second. other investigators have demonstrated SRIF-L1 his-

269 tochemically in cultures of prenatal rat neocortex 7, and in early postnatal rats, in vivo~S. We have combined immunohistochemical examination with R I A measurements. The concordance of the anatomical and biochemical data reported here indicate that immunohistochemical methods can, in this system at least, provide an assay for the level of production, as well as the morphology, of peptidergic neurons. Third, peripheral hormones such as thyroid hormones are likely candidates for constituents of socalled defined culture media in experiments designed to determine the nutritional and hormonal requirements of cells in culture 6,10. Often such analyses rely on experimental end points such as morphologic appearance or survival of cultered cells. Our data suggest that functional parameters can be markedly affected by culture medium components, apparently in the absence of cell death or gross morphological changes. With the possible exception of an apparent reduction in the number or extent of 'background' flat cells in all dishes of brain cells maintained in serum-free conditions, we observed no gross morphologic changes during our experiments. The present results also extend the CNS effects of thyroid hormones to an influence on cerebral cortex somatostatin. It is interesting that sensitivity to thyroid hormones is exhibited by somatostatin levels in a brain region not directly associated with central regulation of anterior pituitary release. The data indicate that the effects of thyroid hormones could be mediated, at least in some instances, by reduced synthesis or release of a neuropeptide. Alternatively, thyroid hormones could act to modulate neurosecretion in general, with somatostatin levels providing a useful marker for such an effect, Furthermore, it is interesting that direct CNS administration of SRIF-L1 and its analogues inhibit sympathetic nervous system activity 4,~2 and influence homeostatic parameters such as thermoregulation 5, both of which are also affected

REFERENCES 1 Ben-Baruch, G., Egozi, Y., Kloog, Y., Mashiach, S. and Sokolovsky, M., Altered ontogenesis of muscarinic cholinergic receptor in mouse brain: effect of L-thyroxine and betamethasone. Endocrinology, 109 (1981) 235-239. 2 Bhat, N. R., Rao, G. S. and Pieringer, R. A., Investigations on myelination in vitro, J. Biol. Chem., 256 (1981) 1167-1171.

by thyroid status. The present data do not permit us to determine whether the observed effects of thyroid hormones pertain to the developing or to the mature nervous system, or possibly, to both conditions. Furthermore, since the possibility cannot be excluded that our observations apply solely to brain cells in vitro, appropriate in vivo paradigms will be necessary to determine the biological significance of our findings. Biochemical analysis of the mechanisms of thyroid hormone action, focusing in particular on the relationships between synthesis, storage and content, degradation, and basal and stimulated release of somatostatin, will also help to clarify the relationship between these two regulatory agents. Nevertheless, the data clearly indicate an interaction between thyroid hormones and brain peptide physiology and point towards potential ways in which the actions of peripheral hormones and central neuropeptides may be integrated. AKNOWLEDGEMENTS We would like to thank Gayle Yamamoto, Tania Burton and Donna Chin for excellent technical assistance; Drs. Nicholas Alexander, Steven Edwards and Thomas Bruhn for helpful discussion, and Dorothy Weigand and Susan McCall for manuscript preparation. This work was supported in part by N I A D D K Grants AM-26741 and AM-20917, N I A A A Grant AA-03504, a grant from the March of Dimes-Birth Defects Foundation, and a Basic Science Research Grant from The Salk Institute. This research was conducted in part by the Clayton Foundation for Research-California Division. W.W.V. and P.E.S. are Clayton Foundation Investigators. R.P. is a recipient of Medical Scientist Training Program Award PHS GM07198.

3 Bhat, N. R., Sarlieve, L. k., Rao, G. S. and Pieringer, R. A., Investigations on myelination in vitro, J. Biol. Chem.. 254 (1979) 9342-9344. 4 Brown, M. R. and Fisher, L. A., Brain peptide regulation of adrenal epinephrine secretion, Amer J. Physiol. l() (1984) E41-E46. 5 Brown, M., Ling, N. and Rivicr, J., Somatostatin-28, somatostatin-14 and somatostatin analogues: effect on thermoregulation, Brain Research. 214 ( 1981) 127-135.

27t~ 6 Brunner, G., Lang, K., Wolfe, R. A., McClure, D. B. and Sato, G, H., Selective cell culture of brain cells by serumfree, hormone supplemented media: a comparative morphological study, Develop Brain Res., 2 (1982) 563-575. 7 Delfs, J., Robbins, R., Connolly, J. L., Dichter, M. and Reichlin. S., Somatostatin production by rat cerebral neurons in dissociated cell culture, Nature (Lond.), 283 (1980) 676-677. 8 Diez-Guerra, J., Aragon, M. C., Gimenez, C, and Valdivieso, F., Effect of thyroid hormones on the malic enzyme activity in rat brain during development, Develop Neurosci., 4 (1981) 130-133. 9 Dozin-vanRoye, B. and DeNayer, P., Nuclear triiodothyronine receptors in rat brain during maturation, Brain Research, 177 (1979) 551-554. 10 Faivre-Bauman, A., Rosenbaum, E., Puymirat, J., Grouselle D. and Tixier-Vidal, A., Differentiation of fetal mouse hypothalamic cells in serum-free medium, Develop. Neurosci., 4 (1981) 118-129. 11 Fellous, A., Lennon, A. M., Francon, J. and Nunez, J., Thyroid hormones and neurotubule assembly in vitro during brain development, Europ. J. Biochem., 101 (1979) 365-376. 12 Fisher, D. A. and Brown, M., Somatostatin analog: plasma catecholamine suppression mediated by the central nervous system, Endocrinology, 107 (1980) 714-718. 13 Grave, G., Thyroid Hormones and Brain Development. Raven Press, New York, 1977. 14 Hamburgh, M., Mendoza, L.A., Bennet, 1., Krupa, P., Kim, Y. S., Kahn, R., Hogreff, K. and Frankfurt, H.. Some unresolved questions of brain-thyroid relationships. In G. Grave (Ed)., Thyroid Hormones and Brain Development. Raven Press, New York, 1977, pp. 49-72. 15 Hartman, B. K., Immunofluorescence of dopamine-/4-hydroxylase: application of improved methodology to the localization of the peripheral and central noradrenergic nervous system, J. Histochem. Cytochem., 21 (1973)312-332. 16 Honegger, P. and Lenoir, D., Triiodothyronine enhancement of neuronal differentiation in aggregating fetal rat brain ceils cultured in a chemically defined medium. Brain Research, 199 (1980) 425-434. 17 Jorgensen, E. C., Thyroid hormone structure-function relationships. In S. C. Werner and S. H, Ingbar (Eds.), The Thyroid, 4th edn.. Harper and Row, Inc. Hagerstown, MD, 1978, pp. 125-138. 18 McDonald, J. K., Parnevelas, J, G., Karamanlides, A. N.. Brecha, N. and Koenig, J. I., The morphology and distribution of peptide-containing neurons in the adult and developing visual cortex of the rat. I. Somatostatin. J. Neurocvtol., 11 (1982) 809-824. 19 Myant, N. B., The role of the endocrine glands in mammalian brain development. In R. Paoletti and A. N. Davison (Eds.), Chemistry and Brain Development, Plenum Press, New York, 1971, pp, 227-238. 20 Oklund. S. and Timiras, P. S., Inlluences of thyroid levels in brain ontogenesis in vivo and in vitro. In G. Grave (Ed:), Thyroid Hormones and Brain Development, Raven Press, New York, I977, pp. 33-48. 21 Oppenheimer, J. H., Thyroid hormone action at the cellular level, Science, 203 (1979) 971-979.

22 Oppenheimer. J. H., Ditlman. W. H.. Schwartz, H. k. and Towle. H. C., Nuclear receptors and thyroid hormone action: a progress report. Fed. Proc.. 38 (1979) 2154-2161 23 Patel, A. J.. Lewis, P. D.. Balazs. R., Bailey, P. and Lai. M.. Effects of thyroxine on postnatal celt acquisition in the rat brain, Brain Research. 172 (1979) 57-72. 24 Patel. A. J.. Smith, R, M . Kingsbury, A. E.. Hunt, A. and Balazs. R.. Effects of thyroid state on brain development: muscarinic acetylcholine and GABA receptors. Brain Research. 198 [19801 389-402. 25 Pelton. E. W. and Bass. N. H.. Adver,~c effects ot excess thyroid hormone on the maturation of rat cerebrum. Arch. Neurol.. 29 (19731 145-150. 26 Peterfreund. R. A.. Somatostatin secrctton trove cultured brain cells: effects of GABA. chotinergic agonists and ovine CRF, Endocrinology, (Suppl.) Ilt) (1982) 139. 27 Peterfreund. R. A, and Vale. W. W,. High molecular weight somatostatin secretion by cultured rat brain cells. Brain Research. 239 (1982/ 463-477 28 Peterfreund. R. A. and Vale. W. W., ()vine corticotropmreleasing factor stimulates somatostatin secretion from cultured brain cells, Endocrinology, 112 [ 1983) 1275-t278, 29 Robbins. R. J.. Sutton. R. E. and Rcichlin. S.. Effects oi neurotransmitters and cyclic AMP on somatostatin release from cultured cerebral cortical ceils, Brain Research. 2M (1982) 377-385. 30 Schmued. I_. C., Swanson. L. W_ and Sawchenko. 1'. E.. Some fluorescent counterstains for neuroanatomical studles. J, Histochem. Cytochem. . 3(1119821 123-128. 31 Seigor, A and Granholm A-C.. Thymxine dependencyof the developing locus coeruleus. Cell 'l'is,sue Res. 220 [ 1981) I 15 32 Shanker. G and Pieringer, R. A.. EIfcct of thyroid h~rmone on the synthesis of sialosyl galactosylceramide (GMaJ m myelinogenic cultures of cells dissociated from embryonic mouse brain. Develop. Brain Res., 6 119831 169-174 33 Singhal. R. L,. Rastogi, R. B and Agarwal. R. A.. Brain biogenic amines in mental dysfunctions attributable to thyroid hormone abnormalities In S, Kumar fed.), Biochemistry of Brain. Pergamon Press Oxford, 1980, pp. 143-184. 34 Smith. R. M . Patel, A. J.. Kingsbur~, A. E., Hunt. A. and Balazs. R.. Effects of thyroid state ~m brain development: /#adrenergic receptors and 5' nucleotidase activity. Brain Research. 19811980) 375-387. 35 Sterling, K , Thyroid hormone action at the cell level part 1, New Engl. J. Med.. 300 ~1979) 117- 123 36 Sterling, K., Thyroid hormone actton at the cell level, part 2. New Engl. J. Med.. 30011979"b 173-177. 37 Valcana. T. and Eberhardt. N. t_... Effect of neonatal hypothyroidism on protein synthesis in the developing rat brain: an open question. In G. Grave (Ed.l, Thyroid Hormones" and Brain Development. Raven Pres,~ New York, 1977. pp. 271-286. 38 Vale. W., Burgus, R. and Guillcmm, R.. On the mechanism of action of TRF: effects of cvctoheximide and actinomycin m the release of TSH stimulated in vitro by TRF and its inhibition bv thyroxine. Neuroendocrinotogy, 3 (19681 34-46.