File Name: SLS1_13.DOC

EXPERIMENT SLS1 BSP13

CYTOCHEMISTRY OF NEURONS

[PIs:]{.underline}

[Russia:]{.underline}

L.M. Gershtein, Brain Research Institute, Russian Academy of Medical
Sciences, Moscow

[Co-Investigators:]{.underline}

A.V. Sergutina, Brain Research Institute, Russian Academy of Medical
Sciences, Moscow

[US:]{.underline}

N. Daunton, NASA Ames Research Center, Moffett Field, California

W. Mehler, NASA Ames Research Center, Moffett Field, California

F. D\'Amelio, NASA Ames Research Center, Moffett Field, California

[Introduction]{.underline}

Biochemical studies of the frontal area of the cerebral cortex of rats
flown on Cosmos-782 \[3\] and Cosmos-936 \[2\] demonstrated a
significant decrease of acetyl cholinesterase (ACE) activity and
sulphydryl group content which was suggestive of a reduced function of
that area of the cerebral cortex in weightlessness. Further detailed
morphological examinations of the somatosensory cortex of rats flown on
Cosmos-1667 \[1, 4\], Cosmos-1887 \[4\] and Cosmos-2044 \[4\] revealed
structural changes of the cortex that varied in their degree. In this
context, a study was performed to identify mechanisms responsible for
adaptive changes in the somatosensory cortex developing in microgravity.
To do this, activities of ACE, monoaminoxidase (MAO) and glutamate
dehydrogenase (GLDH) in the somatosensory cortex and in the head of the
caudate nucleus of the brain of rats flown for 9 days on SLS-1 were
measured.

[Materials and Methods]{.underline}

The frontal portion of the left hemisphere of the rat brain which
incorporates the somatosensory cortex and the head of the caudate
nucleus was dissected from the flight and controls rats (dissected on
the day of recovery, R+0 and C+0, or 9 days after recovery, R+9 and
C+9). The brain fragment was placed into an aluminum foil boat
containing 8% aqueous solution of gelatin and frozen at -150 oC by
keeping in Freon-12 cooled in liquid nitrogen. The frontal fragment of
the left hemisphere was dissected and frozen at the recovery site by
U.S. specialists. The frozen biosamples were shipped to NASA Ames
Research Center where it was kept in liquid nitrogen until shipped to
Moscow. They were shipped to Moscow at -70 oC in Teflon stoppered vials
filled with water ice precooled to -70 oC and then stored in liquid
nitrogen till cryostat sectioning. The frontal sections, 20 mm thick,
that were cut across the somatosensory cortex and the head of the
caudate nucleus in a cryostat at -16 oC, were used to measure ACE
according to Karnovsky and Roots \[6\], MAO according to Glenner et al.
\[5\], and GLDH according to Rubinstein et al. \[7\]. The rate of
histochemical reactions was measured by means of

a LUMAM-IZ cytophotometer at 589 nm for MAO and GLDH and at 488 nm for
ACE. ACE activity was determined in intercellular fibrillar structures
of the III and V layers

of the somatosensory cortex and medium-size neuronal bodies of the head
of the caudate nucleus. MAO activity was determined in intercellular
fibrillar structures of the III and

V layers of the somatosensory cortex of the head of the caudate nucleus.
GLDH activity was measured in the bodies of pyramidal cells of the III
layer and in the bodies of giant pyramidal Betz cells of the V layer, as
well as in medium-size neuronal bodies of the head of the caudate
nucleus. Enzyme activity was expressed as arbitrary units of optic
density (AUOD). The resultant data were treated using the Student-Fisher
test. Histochemical reactions and subsequent cytophotometric analysis
were performed in brain sections of three R+0 rats and four R+9 rats. It
was not possible to prepare sections of two brain samples of R+0 rats
and one brain sample of R+9 rats because the structures were crushed in
the cryostat due to cracks that, evidently, emerged in the course of
freezing.

[Results and Discussion]{.underline}

The R+0 rats did not show any significant changes of ACE and MAO in
fibrillar structures of the III layer of the somatosensory cortex (Table
1). GLDH activity in neuronal bodies was 7% lower; this decrease was not
however significant. ACE tended to decline and MAO decreased by 14% in
fibrillar structures of the V layer of the

somatosensory cortex (Table 2). GLDH in bodies of Betz pyramidal cells
remained essentially unchanged. In the head of the caudate nucleus of
the R+0 rats MAO was

18% lower in fibrillar structures ACE was 28% lower in medium-size
neuronal bodies, whereas GLDH in the same nerve cells did not changes
(Table 3).

ACE, MAO and GLDH did not display any significant changes in fibrillar
structures and neuronal bodies of the III and V layers of the
somatosensory cortex and

the head of the caudate nucleus of the brain of R+9 rats (Tables 1, 2,
3).

The decrease of MAO in fibrillar structures of the V layer of the
somatosensory cortex of rats flown in space for 9 days may point to a
decline in the activity of monaminergic structures of the brain that
modulate Betz pyramidal cells. ACE activity in the above layers of the
somatosensory cortex did not vary substantially. It is very likely that
9-day exposure is too short a time for ACE activity to diminish, as was
the case with ACE level in the frontal cortical area of rats after a
19-day space flight \[3\].

It is known that the caudate nucleus is part of the extrapyramidal
system of the brain which, together with the pyramidal system, is
involved in the realization of motor function, producing an inhibitory
effect on behavioral acts. In this context, diminished activity of ACE
in neuronal bodies of the head of the caudate nucleus after a 9-day
flight can be viewed as evidence of reduced activity of neurons therein
and diminished inhibitory effect of the caudate nucleus on brain
structures. One of the factors responsible for this can be a reduction
of the modulating effect of monaminergic structures which is suggested
by a lowered activity of MAO in fibrillar structures of the head of the
caudate nucleus of R+0 rats.

The above ACE and MAO changes in the somatosensory cortex and caudate
nucleus detected in R+0 rats were, evidently, functional because the
enzyme activity

9 days after recovery were essentially similar to that of the C+0 rats.

Thus, 9-day exposure to weightlessness caused MAO decrease in the V
layer of the somatosensory cortex and the head of the caudate nucleus of
the brain indicating that the modulating effect of monaminergic
structures on those formations diminished in weightlessness. However,
lowered ACE activity in neuronal bodies of the head of the caudate
nucleus pointed to a decline in the functional activity of neurons and,
consequently, a reduction of the inhibitory effect of the caudate
nucleus on globus pallidus, n. ruber, substantia nigra and other
cerebral structures.

[References]{.underline}

1\. Belichenko, P.V. Quantitative Analysis of Dendrite Spines of
Pyramidal Neurons of the V Layer of the Somatosensory Cortex of Rats
Flown on Cosmos-1667. Bull. Exper. Biol., 1988, vol. 105, No. 6, pp.
736-738 (in Russian)

2\. Gazenko, O.G., Dyomin, N.N., Panov, A.N. et al. Neurochemical
Characterization of Rats After Cosmos-936 Space Flight. DAN SSSR, 1979,
vol. 247, No. 2, pp. 510-512 (in Russian)

3\. Gazenko, O.G., Dyomin, N.N., Panov, A.N. et al. et al. Neurochemical
Characterization of Rats During and After Cosmos-782 Space Flight. Kosm.
Biol. Aviakosm. Med., 1979, vol. 13, pp. 22-26 (in Russian)

4\. Dyachkova, L.N. Ultrastructural Characteristics of Plastic Changes
in the Brain Cortex of Rats Exposed to Space Flight. The Physiologist,
1991, vol. 34, No. 1, Suppl., pp. 185-186

5\. Glenner, G.G., Burtner, H.J., Brown, G.W. The Histochemical
Demonstration of Monaminoxidase Activity by Tetrazolium Salts. J.
Histochem. Cytochem., 1957, vol. 5, No. 6, pp. 591-600

6\. Karnovsky, M.J., Roots, L. A. \"Direct-Coloring\" Thiocholine Method
for Cholinesterases. J. Histochem. Cytochem., 1964, vol. 12, No. 3, pp.
219-221

7\. Rubinstein, L.J., Klatzo, I., Miquel, J. Histochemical Observation
on Oxidative Enzyme Activity of Glial Cells in a Local Brain Injury. J.
Neuropathol. Exptl. Neurol., 1962, vol. 21, No. 1, pp. 116-136.

[Summary]{.underline}

Results of cytophotometric and cytochemical examinations of acetyl
cholinesterase, monaminoxidase and glutamate dehydrogenase activity in
the III and V layers of the somatosensory cortex and the head of the
caudate nucleus of the brain of rats flown for 9 days on SLS-1 give
evidence that the exposure diminished MAO in fibrillar structures of the
V layer of the somatosensory cortex and the head of the caudate nucleus
and reduced ACE in neuronal bodies of the head of the caudate nucleus.
These changes can be interpreted as an indication of a decline of 1)
modulating effect of monaminergic structures on the somatosensory cortex
and the head of the caudate nucleus and 2) inhibitory effect of neurons
of the caudate nucleus on globus pallidus, n. ruber, substantia nigra
and other cerebral structures.

[Table 1]{.underline}

Quantitative Cytochemical Analysis of Enzyme Activity in the III Layer
of the Somatosensory Cortex of the Brain of SLS-1 Rats

  -------------- --------------------------------------------------------
                 Rat group

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

  -------------- -------------- -------------- -------------- --------------
  Enzyme         C+0            R+0            C+9            R+9

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

  ------------------- -------------- -------------- -------------- --------------
  MAO activity in     0.237±0.002    0.249±0.02     0.260±0.002    0.261±0.002
  fibrillar                                                        
  structures, AUOD,                                                
  M[+]{.underline}m                                                

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

  ------------------- -------------- -------------- -------------- --------------
  ACE activity in     0.231±0.003    0.234±0.004    0.354±0.004    0.366±0.004
  fibrillar                                                        
  structures, AUOD,                                                
  M[+]{.underline}m                                                

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

  ------------------- -------------- -------------- -------------- --------------
  GLDH activity in    0.277±0.002    0.257±0.006    0.323±0.003    0.316±0.002
  neuronal bodies,                                                 
  AUOD,                                                            
  M[+]{.underline}m                                                

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

[Table 2]{.underline}

Quantitative Cytochemical Analysis of Enzyme Activity in the V Layer of
the Somatosensory Cortex of the Brain of SLS-1 Rats

  -------------- --------------------------------------------------------
                 Rat group

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

  -------------- -------------- -------------- -------------- --------------
  Enzyme         C+0            R+0            C+9            R+9

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

  ------------------- -------------- --------------- -------------- --------------
  MAO activity in     0.287±0.002    0.247±0.002\*   0.310±0.001    0.316±0.002
  fibrillar                                                         
  structures, AUOD,                                                 
  M[+]{.underline}m                                                 

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

  ------------------- -------------- -------------- -------------- --------------
  ACE activity in     0.322±0.004    0.303±0.005    0.519±0.007    0.541±0.007
  fibrillar                                                        
  structures, AUOD,                                                
  M[+]{.underline}m                                                

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

  ------------------- -------------- -------------- -------------- --------------
  GLDH activity in    0.422±0.004    0.404±0.006    0.466±0.004    0.463±0.002
  neuronal bodies,                                                 
  AUOD,                                                            
  M[+]{.underline}m                                                

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

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

[Table 3]{.underline}

Quantitative Cytochemical Analysis of Enzyme Activity in the Caudate
Nucleus

of the Brain of SLS-1 Rats

  -------------- --------------------------------------------------------
                 Rat group

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

  -------------- -------------- -------------- -------------- --------------
  Enzyme         C+0            R+0            C+9            R+9

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

  ------------------- -------------- --------------- -------------- --------------
  MAO activity in     0.246±0.002    0.202±0.003\*   0.279±0.003    0.272±0.003
  fibrillar                                                         
  structures, AUOD,                                                 
  M[+]{.underline}m                                                 

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

  ------------------- -------------- --------------- -------------- --------------
  ACE activity in     1.259±0.012    0.907±0.010\*   1.135±0.011    1.137±0.010
  fibrillar                                                         
  structures, AUOD,                                                 
  M[+]{.underline}m                                                 

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

  ------------------- -------------- -------------- -------------- --------------
  GLDH activity in    0.436±0.003    0.422±0.002    0.527±0.005    0.502±0.006
  neuronal bodies,                                                 
  AUOD,                                                            
  M[+]{.underline}m                                                

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

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