File Name: SLS1_17.DOC

EXPERIMENT SLS1 BSP17

HISTOCHEMISTRY OF THE HYPOTHALAMUS

[PIs]{.underline}

[Russia:]{.underline}

I. B. Krasnov, Institute of Biomedical Problems

[USA:]{.underline}

R. Grindeland, NASA Ames Research Center

P. Sawchenko, Salk Institute, La Jolla, California

W. Veil, Salk Institute, La Jolla, California

[Introduction]{.underline}

Morphological and biochemical analysis of the anterior hypothalamus and
the anterior lobe of the pituitary gland of rats flown on Cosmos
biosatellites and Spacelab-3 revealed inhibition of the synthesis and
excretion of growth hormone and the system controlling these processes
\[1, 4, 5, 6\]. It was also demonstrated that neurons of the arcuate
nucleus (AN) of the hypothalamus synthesizing the growth hormone
releasing factor (GRF) were very sensitive to glutamate that acts as a
neurotransmitter in glutamatergic structures of the brain \[9\] and is,
primarily, formed from glutamine by means of phosphate activated
glutaminase. It is also known that glutamate is a direct precursor

of GABA responsible for the regulation of AN cellular function. Using
qualitative histochemical methods, we previously showed the presence of
glutaminase in neuronal bodies and AN neuropile as well as in the medial
eminence (ME) where endings of axons of AN neurons are located. Having
in view all these observations, we performed a qualitative histochemical
examination of glutaminase activity in the AN and ME of the hypothalamus
of rats flown on SLS-1 for 9 days. The purpose of the study was to
determine the role of enzymes responsible for glutamate synthesis in the
decline of functional activity of structures of the anterior
hypothalamus.

[Materials and Methods]{.underline}

The hypothalamus of rats of the flight group dissected immediately after
recovery (F+0 group), vivarium controls (C+0 group), flight group
dissected 9 days after recovery (F+9 group) and vivarium controls (C+9
group) was removed and placed into 8% aqueous solution of gelatin in
aluminum foil boats. Brain samples were frozen at -150 oC by placing the
boats into Freon-12 precooled in liquid nitrogen. Hypothalamus samples
were dissected and frozen in Freon-12 by a U.S. investigator at the
Columbia recovery site. The frozen samples in a boat were placed into
liquid nitrogen, shipped to NASA ARC and stored there in liquid nitrogen
until shipment to Moscow. The biosamples were transported to Moscow at
-70 oC in stoppered Teflon vials that were preliminarily filled with
water ice cooled to -70 oC . The biosamples were delivered to the
Institute of Biomedical Problems, Moscow, where they were stored in
liquid nitrogen until they were sectioned in a cryostat. Frontal
sections of the anterior hypothalamus, 20 μm thick, were prepared in a
cryostat at -18 oC . Some sections were placed on slide glasses, dried,
and stained for 6 - 8 sec with an alkaline solution of methylene blue to

identify hypothalamus structures. The hypothalamus sections were placed
into cells of a cassette \[3\] and freeze-dried at -32 oC and 2 x 10-3
mm Hg for 16 hours. The freeze-dried sections were stored in cassettes
within an ampoule at -20 oC and 5 x 10-2 mm Hg. The freeze-dried
sections of the hypothalamus were used to isolate single fragments of
the AN and the anterior portion of the ME. This manipulation was
performed by means of microtools \[ 7, 8\] under a binocular microscope
in transmitted light at 20 oC and 50% relative humidity. Each fragment
was weighed by means of a quartz fiber balance \[7, 8\] with a
sensitivity of 182.1 ng/mm. The weight of a single AN fragment was
0.2-0.6 μg and that of the ME was 0.2-1.0 μg. After weighing, some
fragments were placed into an extractor to extract lipids by means of 25
μl of 100o ethanol (applied twice) and hexane with subsequent removal of
the solvents *in* *vacuo* \[2\]. Then the samples were weighed again and
the content of lipids and defatted dry tissue which is essentially
protein was calculated on the basis of the weight difference before and
after extraction.

To measure glutaminase activity \[10\], each fragment was weighed and
placed onto the bottom of a tube (10 x 75 mm); the tubes were put on an
ice bath (+2 oC) and 41.18 μl of incubation media were added. The media
contained, at a final concentration, 50 mM/l Tris.HCl buffer, pH = 8.57;
20 mM/l glutamine; 150 mM/l KH2PO4; 0.6 mM/l EDTA-2Na; 0.014% bovine
serum albumin, and 0.014% Triton X-100. Incubation was carried out at 38
oC for 90 minutes. After incubation the tubes were again placed on an
ice bath, and 20.46 μl 0.7 N HCl was added to arrest the enzymic
reaction. Then the tubes were supplemented with 991 μl of an indicator
reagent containing, at a final concentration, 50 mM/l Tris.HCl buffer,
pH = 8.57; 0.3 mM/l NAD; 0.3 mM/l ADP and glutamate dehydrogenase (from
bovine liver, suspension in 50% glycerol) in the amount required to
oxidize 1 x 10-9 M glutamate in the incubation media at 23-25 oC for 20
minutes. Then the tubes were removed from the ice bath and kept at 23-25
oC for 25 minutes. After this time period fluorescence intensity of
reduced NAD was measured using a fluorimeter EIL-27A with an excitation
maximum at 365 nm and a fluorescence maximum at 460 nm. Glutaminase
activity was expressed as moles of glutamate formed per kg dry tissue
per hour (M/kg dry tissue/hour) or as moles of glutamate formed per kg
defatted dry tissue (M/kg DFDT/hour). Each analytical series included
glutamate references of three concentrations, viz., 0.0248 mM/l, 0.0654
mM/l and 0.195 mM/l , as well as brain homogenate at a dilution of 1 \--
\> 15,000 and 1 \-- \> 40,000. Glutaminase activity was measured in 4-6
fragments of AN and ME of each animal. The data obtained were subjected
to statistical treatment. Examinations were performed using sections of
the hypothalamus of 3 rats from the R+0 group, 4 rats from the C+0
group,

3 rats from the R+9 group, and 3 rats from the C+9 group. It was not
possible to prepare sections of the hypothalamus of the other rats
because it was crushed in the cryostat due to cracks that, evidently,
emerged in the course of freezing.

[Results and Discussion]{.underline}

Immediately after the 9-day flight (R+0 rats) glutaminase activity
decreased in AN by 22.7% (Table 1) and in ME by 30.4% (Table 2) as
compared to the controls, while the ratio of defatted dry tissue and
lipids in the R+0 rats did not change (Tables 1 and 2). In the R+9 rats
glutaminase activity, lipid content and defatted dry tissue in AN and ME
was essentially identical to those in the C+9 rats (Tables 1 and 2).

Diminished activity of glutaminase in AN and ME of the hypothalamus of
the flight rats indicates that glutamate synthesis in AN and ME was
decreased and that decrease in the synthesis of glutamate, which has
exciting influence on GRF synthesizing neurons in AN, may have relation
to lowering the activity of hypothalamic structures participating in the
synthesis and excretion. At the same time the lowered activity of
glutaminase was reversible after return to the Earth. However, further
study is required to identify the role played by this change in the
mechanism underlying regulation the synthesis and excretion of growth
hormone in weightlessness.

[Table 1]{.underline}

Activity of glutaminase, content of lipids and defatted dry tissue in
the

arcuate nucleus of the hypothalamus of rats flown for 9 days on SLS-1

  -------------- ----------------- -------------- -------------- -----------------
  Rat group      Glutaminase       Content of     Content of     Glutaminase
                 activity, M/kg    lipids, %      defatted dry   activity, M/kg
                 dry tissue/hour                  tissue (DFDT), DFDT/hour (M
                 (M                               %              [+]{.underline}
                 [+]{.underline}                                 m)
                 m)                                              

  -------------- ----------------- -------------- -------------- -----------------

  -------------- ----------------- -------------- -------------- -----------------
  C + 0          0.186             32.1           67.9           0.273
                 [+]{.underline}                                 [+]{.underline}
                 0.017                                           0.025

  -------------- ----------------- -------------- -------------- -----------------

  -------------- ----------------- -------------- -------------- -----------------
  R + 0          0.143             32.5           67.5           0.211
                 [+]{.underline}                                 [+]{.underline}
                 0.008\*                                         0.012\*

  -------------- ----------------- -------------- -------------- -----------------

  -------------- ----------------- -------------- -------------- -----------------
  C + 9          0.178             31.9           68.1           0.261
                 [+]{.underline}                                 [+]{.underline}
                 0.014                                           0.020

  -------------- ----------------- -------------- -------------- -----------------

  -------------- ----------------- -------------- -------------- -----------------
  R + 9          0.184             35.2           66.8           0.275
                 [+]{.underline}                                 [+]{.underline}
                 0.008                                           0.012

  -------------- ----------------- -------------- -------------- -----------------

\* p \< 0.05 as compared to C + 0 rats

[Table 2]{.underline}

Activity of glutaminase, content of lipids and defatted dry tissue in
the

medial eminence of the hypothalamus of rats flown for 9 days on SLS-1

  -------------- ----------------- -------------- -------------- -----------------
  Rat group      Glutaminase       Content of     Content of     Glutaminase
                 activity, M/kg    lipids, %      defatted dry   activity, M/kg
                 dry tissue/hour                  tissue (DFDT), DFDT/hour (M
                 (M                               %              [+]{.underline}
                 [+]{.underline}                                 m)
                 m)                                              

  -------------- ----------------- -------------- -------------- -----------------

  -------------- ----------------- -------------- -------------- -----------------
  C + 0          0.239             31.7           68.3           0.349
                 [+]{.underline}                                 [+]{.underline}
                 0.024                                           0.035

  -------------- ----------------- -------------- -------------- -----------------

  -------------- ----------------- -------------- -------------- -----------------
  R + 0          0.169             30.6           69.4           0.243
                 [+]{.underline}                                 [+]{.underline}
                 0.013\*                                         0.018\*

  -------------- ----------------- -------------- -------------- -----------------

  -------------- ----------------- -------------- -------------- -----------------
  C + 9          0.225             32.2           67.8           0.331
                 [+]{.underline}                                 [+]{.underline}
                 0.017                                           0.025

  -------------- ----------------- -------------- -------------- -----------------

  -------------- ----------------- -------------- -------------- -----------------
  R + 9          0.211             31.1           68.9           0.306
                 [+]{.underline}                                 [+]{.underline}
                 0.011                                           0.015

  -------------- ----------------- -------------- -------------- -----------------

\* p \< 0.05 as compared to C + 0 rats

[References]{.underline}

1\. Alekseev, E. I., Plakhuta-Plakutina, G. I. Morphofunctional State of
the Adenohypophysis, Thyroid and Parathyroid Glands in Cosmos-1667 Rats
After a 7-Day Flight. Abstr. XX Meeting of the Intercosmos Joint Working
Group on Space Biology and Medicine. Berlin, GDR, 1987, vol. II, p. 218
(in Russian).

2\. Krasnov, I. B. Lipid Extraction from Single Isolated Nerve Cells.
Cytol., 1973, vol. 25, No. 12, pp. 1535-1539 (in Russian).

3\. Krasnov, I. B. Gravimetric Analysis of Lipids Content in the
Neuronal Cytoplasm. Cytol., 1984, vol. 26, No. 11, pp. 1327-1331 (in
Russian).

4\. Alekseev, E. I. Morphological Analysis of Pituitary Somatotrophs in
Rats Flown on Cosmos Biosatellites. Abstr. XII Annual Meet. IUPS
Commission on Gravitational Physiology. Leningrad, USSR, 1990, p. 3.

5\. Grindeland, R., Hymer, W. C., Farrington, M. et al. Changes in
Pituitary Growth Hormone Cells Prepared from Rats Flown on Spacelab-3.
Amer. J. Physiol., 252 (Regulatory Integrative Comp. Physiol., 21),
1987, R209-R215.

6\. Grindeland, R., Hymer, W.C., Sawchenko, P. E. et al. Growth Hormone
Physiology of Rats After Space Flight. Abstr. Intern. Symp. Cosmos
Biosatellites. Leningrad, USSR. Moscow, 1991, pp. 195-196.

7\. Lowry, O. H. The Quantitative Histochemistry of the Brain. I.
Histological Sampling. J. Histochem., Cytochem., 1953, vol. 1, No. 6,
pp. 420-428.

8\. Lowry, O.H. Microanalysis for Histochemical Purposes. In: II Intern.
Congr. Histo- and Cytochem., Frankfurt a/Main, 1964, pp. 62-69.

9\. Millard, W. G., Martin, G. G. B., Auded, G. et al. Evidence that
Reduced Growth Hormone Secretion Observed in Monosodium Glutamate
Treated Rats is the Result of the Deficiency in Growth Hormone Releasing
Factor. Endocrinol., 1982, vol. 100, No. 2, pp. 540-550.

10\. Wiet, G. J., Godfrey, D. A., Ross, C. D., Dunn, J. D. Quantitative
Distribution of Aspartate Aminotransferase and Glutaminase Activities in
the Rat Cochlea. Hearing Res., 1986, vol. 24, No. 1, pp. 137-150.

[Summary]{.underline}

Using quantitative histochemical methods, activity of glutaminase, the
key enzyme of glutamate synthesis in the nervous tissue, as well as the
content of lipids and defatted dry substance were measured in single
fragments of freeze-dried sections (20 microns thick) of the arcuate
nucleus (AN) and medial eminence (ME) of the hypothalamus of rats flown
for 9 days on SLS-1. The weight of single AN and ME fragments dissected
from freeze-dried sections was 0.2 to 1.0 mg. It was found that
glutaminase activity in AN decreased by 22.7% and in ME by 30.4%, while
the proportion of lipids and defatted dry tissue remained unaltered.
Having in view reported data about high sensitivity of
somatoliberin-containing neurons of the AN to glutamate, it can be
suggested that glutamate may be involved in the regulation of growth
hormone excretion.