File Name: SLS1_11.DOC

EXPERIMENT SLS1 BSP11

MORPHOLOGY OF NEURONS

[PIs]{.underline}

[Russia:]{.underline}

T. A. Leontovich, Brain Research Institute, Russian Academy of Medical
Sciences, Moscow

[USA:]{.underline}

O. Lowry, Medical School, Washington University, Saint-Louis, Missouri

[Co-Investigators:]{.underline}

[Russia:]{.underline}

P. V. Belichenko, Brain Research Institute, Russian Academy of Medical
Sciences, Moscow

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

M. A. Makhanov, Brain Research Institute, Russian Academy of Medical
Sciences, Moscow

[Introduction]{.underline}

Morphological evaluation of brain neurons of animals exposed to space
flight

is required to gain an insight into mechanisms underlying adaptive
responses to weightlessness. Previous studies \[1 - 4\] demonstrated
consistent changes in morphometric parameters of the geometry and
orientation of dendrites of neurons of

medulla oblongata of rats: giant multipolar neurons (GMN) of the giant
cell nucleus of

the reticular formation (RF) of medulla oblongata after a 7-day
Cosmos-1667 flight, rostral nucleus of the RF of pons varolii of rats
after a 14-day Cosmos-1887 flight, and the giant cell nucleus of the RF
of medulla oblongata after a 14-day Cosmos-2044 flight. The present
study yielded morphometric data of dendrite geometry of GMN of the giant
cell nucleus of RF of medulla oblongata of rats flown for 9 days on U.S.
SLS-1 and dissected 2-3 hours or 9 days after recovery.

[Materials and Methods]{.underline}

Brain stem samples of male rats (Sprague-Dowley) from the basal control
group (BC, 5 rats), vivarium control group (V+0, 5 rats), flight group
dissected on the recovery day (R+0, 5 rats), flight group dissected 9
days after recovery (R+9, 5 rats) and vivarium control group (V+9, 5
rats) were examined.

Rats were decapitated, brain fragments isolated and fixed by U.S.
investigators

at the Columbia recovery site. Further brain fragment fixation,
osmification, silver impregnation according to the method of Golgi (in
the Brain Research Institute modification), and embedding in celloidin
were performed by a Russian investigator at NASA Ames Research Center.

Within 3 minutes after decapitation a frontal block of the right brain
stem was dissected. Each isolated fragment was placed into a dark glass
stoppered vial containing 50 ml of 3% K2Cr2O7 in 1% aqueous solution of
Na2SO4 . 10 H20 and allowed to stand in the darkness for 72 hours at +20
oC. Then each fragment was transferred into a dark glass vial containing
50 ml of 0.2% osmium tetroxide in 3% aqueous solution of potassium
bichromate and allowed to stand in a thermostat for 48 hours at +37 oC.
After that each brain fragment was placed into a Petri dish containing
50 ml of 1% aqueous solution of silver nitrate, washed quickly and
placed into a dark glass vial containing 50 ml of 1% aqueous solution of
silver nitrate and allowed to stand in the darkness for 7 days at +20
oC.

After impregnation brain samples were dehydrated in ethanols of
ascending concentration (70o, 80o, 90o for 15 minutes at each; 96o, 100o
for 1 hour at each; 100o ethanol - diethyl ether mixture (1: 1) for 30
minutes at +20 oC), placed into celloidin

solutions of ascending concentration (2.5% for 1 hour; 5% for 12-16
hours), and embedded in 10% celloidin solution. 72 hours later blocks
were placed into a desiccator containing 40-60 ml chloroform to allow
the celloidin to become densely packed. The densely packed celloidin
blocks were shipped to the Institute of Brain Research, Moscow. Each
celloidin block was put onto a separate wooden block of 1 cm x 1 cm x 1
cm and

2-3 ml 10% celloidin were placed on the celloidin block, and the wooden
blocks coupled with celloidin blocks were put into an desiccator
containing 10 ml chloroform for 1-2 hours. Then the block was fixed in a
microtome, and 150 μm sections were made with a knife (employed for
celloidin cutting) and placed into a Petri dish containing 98o ethanol.
After that the sections were transferred from 98o ethanol to a cover
glass, embedded in Canadian fir balsam and allowed to stand for 1 - 2
months to allow the balsam to solidify.

Using a drawing unit attached to an Ortolux microscope (Leitz, FRG),
neurons were outlined from histological preparations at a magnification
of x400. By means of a digitizer a graphic drawing of the neurons was
obtained. Morphometric analysis of the geometry of dendrites of GMN of
the RF was performed according to the method developed by T.A.
Leontovich \[5\] with the aid of an IBM PC software custom built by

A. A. Fedorov. All parameters were entered to a database software
developed by A. A. Makhanov. The results were represented as the mean
(and a standard deviation) and their significance was determined using
the Mann-Whitney test.

[Results and Discussion]{.underline}

Examination of histological preparations of rat brains treated according
to the method of Golgi gave evidence that the biosamples were dissected
thoroughly, all brain structures were well preserved, and that neuronal
bodies and dendrites were adequately impregnated with silver in all
animal groups.

The effect of space flight factors on rat brain stem structures was
investigated

in terms of morphometric parameters of the geometry of GMN of the RF.
These neurons are mainly located in the medial compartments of the RF
and have very long (up to 1 mm) dendrites radially extending toward
sensory nuclei of cranio-cerebral nerves. Our previous observations
suggest that these neurons are cells responsible for automatic motor
acts. The GMN of the RF of medulla oblongata examined in this study
normally direct a significant number of their dendrites toward
vestibular sensory nuclei. This leads us to believe that they are
directly involved in motor reflexes based on vestibular sensory
information.

Table 1 presents morphometric parameters of GMN of the RF of medulla
oblongata of rats flown on SLS-1 (R+0 and R+9 rats) and used in
ground-based control

experiments (V+0 and V+9). Morphometric parameters describe the area of
the profile field of the neuronal body (Scl), number of primary
dendrites (d), their total length (Ld) and geometry: mean branching of
dendrites (Ad) total branching of the cell (Ac), number of foci of the
maximum branching of dendrites within the dendrite territory (FMB),
i.e., they give a full characterization of a neuron.

With respect to all these structural parameters no significant
differences were detected between: 1. neurons of R+0 and V+0 rats and 2.
neurons of R+9 and V+9 rats. However, significant differences were found
between neurons of R+0 and R+9 rats in terms of the mean dendrite
branching which was higher in the R+0 rats (p = 0.04) and the number of
dendrites which was higher in the R+9 rats (p = 0.006) (Table 1). The
lack of significant differences in the above dendrite parameters between
the flight and control rats was also observed in our previous study of
the geometry of neurons of the same reticular magnocellular nucleus of
the reticular formation of medulla oblongata after a 7-day Cosmos-1667
flight \[1, 2\]. However, the flight rats showed an altered orientation
of dendrites of GMN of the reticular formation nucleus: the amount of
dendrites directed toward vestibular nuclei decreased, whereas that of
dendrites going in other directions increased in the flight rats.

At the same time, our investigation of GMN of the RF of rats exposed to
longer 14-day flights (Cosmos-1887 and Cosmos-2044) demonstrated
significant differences between the flight and control rats in terms of
dendrite orientation and structural parameters: the total cell
branching, number of maximal branching foci and volume of

the dendrite territory increased. These findings suggest that during a
short-term (7 days) exposure these stem neurons undergo dendrite
rearrangement to meet the requirements of a qualitatively altered flow
of sensory information, whereas during a longer-term exposure (14 days)
they develop structural changes of compensatory nature.

In this context, it was very interesting to perform a comparative study
of GMN and neurons of a different type, i.e., pyramidal neurons of the
III layer of the visual cortex of the same rats (see SLS-1-17 Final
Science Report). In relation to their physiology, pyramidal neurons are
actively involved in compensatory processes in microgravity. It was
demonstrated that these associative cortical neurons responsible for
intracortical connections developed changes not only in the orientation
of selected dendrite branches but also in structural parameters of
dendrites as early as after 9 days of flight. The specific feature of
this morphologically homogeneous type of pyramidal neurons was that
dendrite parameters altered only in some cells, which were evidently
involved in compensatory processes, whereas the same parameters of other
pyramidal neurons remained unchanged.

A study of gigantic multipolar neurons of medulla oblongata
participating in motor automatic acts and vestibular reflexes of rats
flown for 9 days on SLS-1 in comparison with those of rats flown for 7
and 14 days on Cosmos-1667 \[1, 2, 4\], Cosmos-1887 \[3, 4\] and
Cosmos-2044 helped identify time course variations in the structure of
those neurons in microgravity. The study demonstrated stability of the
structural parameters of dendrites of gigantic multipolar neurons in
short-term flights

(7-9 days), which led only to a re-orientation of dendrites toward other
afferent inputs, and structural changes in longer-term flights (14
days). The SLS-1 study also showed greater stability and lower
adaptability of older stem structures and a significantly higher
adaptability of pyramidal neurons of associative layers of the
neocortex, i.e. pyramidal neurons of the III layer of the visual cortex.
Those latter seem to provide compensatory functions, probably, from very
early stages of animal exposure to microgravity. In summary, the SLS-1
study demonstrated structural foundations underlying changes and
compensatory functions of the brain in space.

At the same time, this study showed significant differences in the
number and mean branching of dendrites between R+0 and R+9 rats, which
pointed to structural rearrangement of dendrite processes of GMN that
occurred at 0 g and 1 g. However, within a short flight period the
rearrangement could not reach a degree detectable through comparison
with vivarium controls.

This study also showed significant differences in terms of the number (p
= 0.03) and mean branching (p= 0.004) of dendrites and total neuronal
branching (p=0.03) (Table 1) between V+0 and V+9 rats, as well as
significant differences in terms of the number and mean branching of
dendrites between R+0 and R+9 rats. These findings can hardly be
explained at present. In order to give them an adequate interpretation,
it is necessary to know all the details of V+0 and V+9 rat handling. Our
earlier investigations of neuronal structures of rats flown in space did
not provide similar data. This raises the question as to whether SLS-1
control rats were exposed to other effects.

[Summary]{.underline}

Morphometric investigation of dendrite geometry of giant multipolar
neurons of nucleus reticularis gigantocellularis of medulla oblongata of
rats flown for 9 days on SLS-1 did not reveal significant differences
between flown and ground-based control animals. However, significant
differences in the number and mean branching of dendrites between R+0
and R+9 rats suggested structural rearrangement of the dendrite tree of
neurons which developed during and after flight. Comparison of those
findings with the data obtained during similar studies in Cosmos-1667,
Cosmos-1887 and

Cosmos-2044 flights helped identify time course variations of the
dendrite tree of gigantic multipolar neurons of the reticular formation
at different stages of animal adaptation to microgravity.

[References]{.underline}

1\. Belichenko, P.V., Leontovich, T.A. Morphometry of Giant Multipolar
Neurons of the Reticular Formation of the Brain Stem of Rats Flown on
Cosmos-1667. Bull. Exper. Biol., 1989, vol. 106, No. 5, pp. 618-620 (in
Russian).

2\. Belichenko, P.V. Morphology of Giant Multipolar Neurons of the
Reticular Formation of the Brain Stem of Rats Flown on Cosmos
Biosatellites. Abstr. Intern. Symp. Cosmos Biosatellites,
Leningrad-Moscow, 1991, pp. 19-20 (in Russian).

3\. Belichenko, P.V., Leontovich, T.A. Study of Giant Multipolar Neurons
of the Reticular Formation of the Brain Stem of Rats after a 14-Day
Space Flight. Aerospace & Environ. Med., 1992, vol. 26, No. 5-6, pp.
24-27 (in Russian).

4\. Belichenko, P. V., Makhanov, M. A., Fedorov, A. A., Krasnov, I. B.,
Leontovich, T. A. Effects of Space Flight on Dendrites of Neurons of the
Rat Brain. The Physiologist, 1990, vol. 33, No. 1, Suppl., pp. S12-S15

5\. Leontowitsch, T.A. Methodik zur quantitativen Beschreibung
subcorticaler Neurone. J. Hirnforschung, 1973, vol. 14, No. 1/2, pp.
59-73.

[Table 1]{.underline}

Morphometric Parameters of Giant Multipolar Neurons of the Giant Cell
Nucleus

of the Reticular Formation of Medulla Oblongata of Rats Flown on SLS-1

During Flight and Recovery

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

Parameter, Animal group

unit
\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

R+0 V+0 R+9 V+9

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

n 44 27 24 31

d 7.7 (2.3) 8.1 (1.8) 9.4 (2.0) 9.3 (1.9)

dl 1.3 (1.3) 1.4 (1.1) 1.9 (1.2) 1.8 (1.4)

Bd 18.8 (5.1) 19.7 (5.4) 19.2 (4.6) 18.0 (4.9)

Scl, μm2 2470 (960) 2924 (1099) 2842 (909) 3428 (1085)

Ad 2.8 (1.0) 2.8 (0.8) 2.4 (1.1) 2.2 (0.5)

Ac 21.0 (7.0) 22.3 (6.9) 21.6 (6.0) 20.1 (5.7)

FMB 1.7 (1.3) 1.7 (1.3) 1.6 (1.7) 1.1 (0.9)

Fdt 2.0 (1.7) 2.1 (1.6) 2.0 (2.1) 1.4 (1.1)

Ld, μm 4125 (993) 4132 (1116) 3647 (798) 3795 (952)

\--

x FMB 0.3 (0.3) 0.3 (0.2) 0.3 (0.3) 0.2 (0.1)

h, μm 113 (8) 107 (6) 106 (4) 106 (9)

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--

Designations:

n - number of cells taken under examination

d - number of primary dendrites

dl - number of cut primary dendrites

Bd - number of free terminals of all dendrites

Scl, μm2 - area of the profile field of the neuronal body

Ad - mean dendrite branching

Ac - total cell branching

FMB - number of foci of the maximal branching of a dendrite

Fdt - number of foci of the maximal branching of a dendrite in a
dendrite

territory

Ld, μm - total dendrite length

\-\-\--

x FMB - mean number of foci of the maximal branching per dendrite

h, μm - section thickness

The values are given as means with standard deviations in parentheses.

\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--