Neuroendocrinology 28: 371-376 (1974)

Subcellular Distribution of Corticotropin-Releasing Factor in the Medio-Basal Hypothalamus of the Rat1 B. Briaud, A. Enjalbert, C. Mialhe and C. Kordon Institut de physiologie, Université Louis-Pasteur, Strasbourg, and Unité de neuroendocrinologie de l’Inserm U-159, Paris

Key Words. Hypothalamus • CRF • Subcellular distribution

Although the pituitary adrenocorticotropic hormone (ACTH)-releasing process was the first thought to be controlled by a hypotha­ lamic neurohumoral substance [8, 17], identi­ fication of this factor, called CRF, has not yet been achieved [18]. Recently, several papers have appeared concerning subcellular distribution of neuro1 This work was supported by the ‘Centre national de la recherche scientifique’ (CNRS, ERA 178) and by the ‘Institut national de la santé et de la recherche médicale (INSERM, U-159). Received: July 3, 1978 Accepted after revision: November 11, 1978

hormones in the hypothalamus (LHRH: Ra­ mirez el al. [16]; TRH: Barnea et al. [1]; SR IF: Epelbaum et al. [6]; P IF : Enjalbert et al. [5], All these peptides were clearly shown to be concentrated in nerve endings. Because of the presence of a substantial CRF activity in the synaptosomes from the mediobasal hypo­ thalamus (MBH) [2, 15], it was of interest to quantify this activity and to determine whether CRF has the same distribution as chemically characterized neurohormones. We, therefore, proposed to study the dis­ tribution of CRF in the subcellular fractions of the MBH of the rat, taking advantage of the fact that under certain conditions, isolated

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Abstract. Subcellular fractionation of the mediobasal hypothalamus (MBH) and frontal cerebral cortex was performed by differential and discontinuous sucrose gradient centrifuga­ tion. Corticotropin-releasing factor (CRF) activity of the different fractions was evaluated by bioassay. Significant CRF activity was found in acidic extracts of the MBH but not of the cerebral cortex. About 80% of the MBH effect on adrenocorticotropic hormone release was re­ covered in the crude mitochondrial pellet (P2) which contains synaptosomes. After further fractionation, distribution of CRF activity paralleled that of lactate dehydrogenase activity, a marker of the soluble cytoplasm. It is concluded that most CRF in the MBH is located in nerve endings as already shown for several other neurohormones.

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nerve endings (synaptosomes) could be ob­ tained [20].

Materials and Methods Male Wistar rats weighing about 200 g were kept at 26 °C with lights on from 06:00 to 20:00 h and fed ad libitum. They were housed 2 or 3 per cage for at least 24 h before the experiments. Animals were killed by decapitation between 08:30 and 09:30 h. After rapid removal of the brain, the hypophysial anterior lobes (AL) were removed and kept at room temperature in Krebs-Ringer bicarbo­ nate buffer (pH 7.4) containing 0.2% glucose and 0.1% BSA (KRA). MBH (including the median emi­ nence and the medial and posterior part of the arcuate nucleus) were transferred to a 2-ml potter containing 0.32 M sucrose (50 /d/hypothalamus) and maintained on ice. The subcellular fractionation of MBH was carried out according to Whittaker [20]. Homogenization of the tissue in 0.32 M sucrose results in the formation of closed vesicles from nerve endings (synaptosomes). From the homogenate (IT; low speed supernatent), we first obtained a high speed supernatent (S2) and a

crude mitochondrial pellet (P2, containing synapto­ somes, mitochondria and smaller membrane debris). The synaptosomes were partially purified by a sub­ sequent fractionation of P2 on discontinuous sucrose gradient. The fractions obtained were: myelin (A), synaptosomes (B), mitochondria + synaptosomes (Q . Portions of each fraction were reserved for determina­ tion of proteins [ 12] and lactate dehydrogenase (LDH). The latter was used as a cytoplasmic marker to indi­ cate the presence of synaptosomes [9]. All the fractions were extracted with 0.1 N HC1 and frozen until assays. CRF activity was determined in vitro using rat anterior pituitaries, as described elsewhere [4]. The response parameter for CRF activity was indicated by the amounts of ACTH released, which were deter­ mined using isolated adrenal cells, as described by Sayers et at. [19]. Results were analyzed using either the Student’s t test for comparison of 2 means (table I) or variance analysis followed by a factorial analysis for com­ parison of S2 and P2 CRF activities (4 point assay method; fig. 1) [11],

Results MBH contained large amounts of CRF ac­ tivity. MBH homogenate (H) strongly stim-

Table I. Subcellular distribution of CRF activity in MBH and cerebral cortex CRF Activity

A g /E q l

100.0 20.8 82.4

318.2±39.0 133.8±22.5 63.8 ±15.9

324.0±92.2 100.0 ±37.0 138.2 ± 4 6 .1

8.5 8.2 0

339.0 ±33.4 134.7 ± 7.7 80.1 ± 5.1

359.3 ± 66.1 82.4 ±17.4 129.3 ±23.7

% increase vs. MBH

MBH (0.5 Eq)

H S2 P2

203.7 ± 32.4 1,144.1 ±121.8* (12) 399.1 ± 31.2* (12) 978.6± 84.9* (12)

Cortex (1 Eq)

H S2 P2

283.8 ± 35.3(8) 281.3± 78.8(8) 182.9± 21.5(8)

Proteins,

LDH Activity, mU/Eq1

ACTH released, ¿í U/60 min/mg Prot

Results given as mean ± SEM of ACTH released from hypophysial AL in vitro. Three experiments were performed. Number of assays in parenthesis. 1 Values are mean ± SEM of 3 fractionations. * p < 0.01, Student’s t test.

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Control

Subcellular Distribution of Hypothalamic CRF

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Fig. 1. Dose-dependent effect of MBH S2 and P2 on ACTH release from incubated AL. Results are given as the mean ± SEM of 8 determinations from 2 independent experiments. Results of statistical anal­ ysis (4-point assay) are shown in the inset (NS = not significant; HS = highly significant; linear regression: F j5 = 10.55, p < 0.01; S2 vs. P2: F |5 = 21.4. p