Development and performance of respirometers for biorhythmic studies of gravity effect

Heberlein, P. J.

Life, J. S.

English

NASA Technical Reports Server

6 March 1969
4
TECHNICAL STATUS REPORT
Contract No. NASw-870
Period 4 November 1968
to 4 February 1969
Support of the work reported herein has
been provided by the Biosciences Program
Division, Office of Space Sciences and
, Applications, Headquarters, National
Aeronautics and Space Administration
d
Prepared for: National Aeronautics and
Space Administration (SB)
Washington, D.C. 20546
Prepared by: Paul J. Heberlein
Project Manager (Phase I)
Jeffry S. Life
Project Manager (Phase II)
SPACE /DEFENSE CORPORATION
1600 North Woodward Avenue
Birmingham, Michigan 48011
• Telephone: (313) 647-1304
https://ntrs.nasa.gov/search.jsp?R=19690011547 2020-03-12T07:31:56+00:00Z
E4
TABLE OF CONTENTS
Page
LIST OF FIGURES iii
1.0 BACKGROUND INFORMATION 1
2.0 PROGRAM STATUS" PHASE I 2
2oi General 2
2o2 Technical 2
2°3 Plans for the Coming Period 6
3.0 PROGRAM STATUS: PHASE II 6
3.1 General 6
3.2 Technical 7
° 3.3 Plans for the Coming Period i0
, ii
LIST OF FIGURES
Figure Number Page
1 Drawing S/D 121-300A 5
2 Labyrinthectomized Fish 9
Swimming Inverted
' iii
1.0 BACKGROUND INFORMATION
This document reports progress on tasks defined under
Contract No. NASw-870, Modification No. 6. Under this agree-
ment, Space/Defense Corporation (herein after "the contractor")
has continued to develop and evaluate the performance of single
and multi-specimen respirometers. The ultimate goal of this
program is to provide instruments suitable for performing
research concerned with biorhythms and the mechanisms respon-
sible for the establishment and control of observed rhythmic
activity.
The configuration of these instruments is specifically
oriented toward the conduct of biological rhythmicity studies
in the space environment. The potato tuber has been chosen as
the experimental subject on the basis of the relative simpli-
city in the basic requirements for the maintenance of a viable
organism over extended periods, and secondly, because of a
great mass of baseline data already collected from this
organism in the earthbound environment.
The program thus far has been responsible for the success-
ful development of single-specimen respirometric systems
selected for flight in the Apollo Applications Program. Cur-
rentl_ development work focuses on multi-specimen systems more
suitable for other potential space experiment applications.
The progress of this work is described under paragraph 2.0,
below.
In addition to these biorhythmic studies, a concurrent,
second phase effort has been initiated to establish the feasi-
bility of developing an experimental population of fishes which
can be abnormally oriented relative to earth gravity, with the
ultimate goal of relating the role of gravity to the regulation
of biological systems. The progress of this program is reported
in paragraph 3.0, below.
2.0 PROGRAM STATUS: PHASE I
2.1 General
During this reporting period, all respirometric
development programs described in the contract are fully active.
Further baseline data has been collected from the single speci-
men units and final assembly of the two multi-specimen has been
completed. Continued problems in regulator hysteresis have, led
to a concerted effort to solve this persistent problem.
2.2 Technical
2.2.1 Single Specimen Respirometer
The single specimen respirometers have opera-
ted continuously for more than i0 months without breakdown.
The tubers had to be replaced twice and the scrubbing units
replaced five times over this period. A new specimen collection
scheme has resulted in maintaining an active organism in the
respiration units for four months or longer. Mechanical
malfunctions have not been observed except for those periods
when the scrubbing of CO2 was appreciably impaired due to
depletion of the scrubbing entity. At this time hysteresis in
the second stage regulatorbecomes a major factor which perturbs
the data appreciably. Performance of the new solenoid valve
has been excellent with no valve malfunction occurring during
the reporting period. The systems are at present closed down,
not because of malfunction, but because the system is regarded
as fully developed.* Furthermore, the amount of data being
generated defies hand-analysis, considering the resources of
Contract NASw-870. However, data collection and reduction
**
(possibly by EDP modes) will be re-instituted soon.
2.2.2 Collaboration with Other Experimental
Activities
During the reporting period active collabora-
tion with the Ames Research Center BIOSATELLITE program group
was maintained. Data and drawings were supplied and hardware
was loaned to NASA-ARC and their contractors, TRW Systems,
Inc., and Northrup Aviation, Inc. In addition, the contractor
participatedin a symposiumat NASA-ARC sponsoredby Head-
quarters, NASA, OSSA, to discuss configuration of a post-AAP
manned biological laboratory and implicating potato respiro-
metric biorhythm experiments. All evidence indicates acceptance
of single or multi-specimen units for flight aboard these
vehicles.
With the exception of the problem of second stage
hysteresis, discussed elsewhere herein.
**
Under the AAP Flight Hardware Contract,
successor to NAS 9-7172.
2.2.3 Multi-Specimen Unit Effort
The second generation unit was assembled and
put on bench test. Data from bench tests indicated that all
systems were successfully operative, except that the second-
stage regulators, during low demand periods, demonstrated the
characteristic hysteresic behavior, perturbing the data quality.
In the past, our approach to this intractable problem had been
to redesign the deformable ("soft seat") seal used to assure
the tight closures needed for high accuracy. Changes in seal
configuration and seal materials had been helpful, but had not
solved the problem to our satisfaction. Accordingly, when a
final assault on the problem was planned we decided to abandon
the "soft seat" concept and take a "hard seat" (metal to metal)
approach. We recognized the problems inherent in this approach,
but reasoned that if the regulator orifice could be kept small
enough and the surfaces smooth enough, the problems might be
avoidable. Accordingly, a design effort was launched, which
has resulted in the configuration shown in Drawing S/D 121-300A,
as Figure io As pressure at "A" decreases due to potato res-
piration, the constant pressure aneroid bellows "B" expands,
pushing plate "C" up, bearing on rod "D", displacing tunsten
carbide valve ball "E", (restrained in place by counter spring
"F"). This allows oxygen under pressure to flow from reser-
voir "G", past the valve ball, rod, plate and bellows to "A"o
The critical interface is the metal-to-metal seal at "H"
where the valve ball seats itself. If lapping tolerances at
"H" are good enough and ball surface smoothness at "E" are
satisfactory, no difficulties with this precision, low-flow
4

regulator should be encountered° Fabrication of bench models
has begun°
2°3 Plans for the Coming Period
Fabrication, assembly and test of the "hard seat" regu-
lator will be accomplished° Performance at low flows will be
compared to the soft seat device°
3.0 PROGRAM STATUS: PHASE II
3ol General
To study the role gravity plays in regulating certain
biological systems, teleost fishes were chosen because their
orientation, relative to gravity, could be altered for pro-
longed periods of time° These lower vertebrates orient to
gravity through integrating stimuli of external (visual) and
internal (vestibular) origin° By surgical elimination of inter-
nal stimuli and altering the external cue, we hypothesized that
this animal could be made to swim consistently in different
orientations relative to the earth's gravitational force field0
thereby providing a usef_l hool in studying the role gravity
plays in neurohypophyseal hormone regulation°
Once fish can be made to swim at predetermined axes
relative to gravity it is desired to determine the effects these
altered orientations may have on the hypothalomo-hypophyseal
system. The accurate assay of endogenous arginine-vasotocin
in tissue sections taken from the hypothalamus, infundibulum
and neurohypophysis of control and experimental fish must there-
fore, also be undertaken° To do this the following events must
occur: arginine vasotocin m,_stbe synthesized; antibodies to
the exogenous hormone developed_ cross-reaction between endoge-
nous hormone and exogenous hormone antibodies shown; and quanti-
tative measurements made of changes in elaboration and/or
transport of endogenous hormone in both control and experimental
fishes.
As of the start of the reporting period we had
successfully synthesized arginine vasotocin and have had
limited success in inducing fish to persistently swim at altered
angles relative to gravity° During the reporting period we con-
tinued work in both biochemical and surgical areas and the con-
. siderable progress is described below°
3.2 Technical
3o2ol Development of the Test Animal
During this reporting period we have continued
work with the angel fish (_um scalare). Surgical pro.=
cedural problems have been worked out with the assistance of
Dr. Jelle Atema (of the Ur_iversityof Michigan group working
under Dr. Rudolf Jo vonBaum_a2ten)o The removal of labrin-
thine structures is performed by making a small incision 3 mm
behind each eye in fish anesthetized with MS222, and carefully
removing the vestibular structures with dissecting forceps°
7
The entire procedure is done with the aid of a dissecting
microscope° Within minutes the fish is swimming in a tumbling
manner, Completely disoriented° This behavior continues until
the third day, when the fish begins orienting toward the single
bright light illuminating the otherwise darkened aquarium° It
is at this time the operated fish also begins eating and inter-
acting with other fish in the aquarium° All behavior appears
normal except for the orientation of the operated fish. As the
light is incrementally moved around to the bottom of the aquariuma
the operated fish again demonstrates disoriented swimming
behavior, but soon "locks on" the light and swims inverted.
The unoperated fish, of course, do not respond. Figure 2
illustrates the behavior of an operated fish continuously
orienting to a light cue rather than gravitational stimuli° A
labyrinthectomized fish is at center. Note the inverted posi-
tion, relative to his unoperated fellows at upper left and upper
right.
3.2°2 Biochemical Effort
During the reporting period a second synthesis
of arginine-vasotocin was successfully performed in our labora-
tory, using the Merrifield technique° The derived material is
highly active on the basis of preliminary assays, showing abo_at
50 times the biologic activity of the oxytocin standard. Puri-
fication procedures have been devised and purification of
synthesized material continues° A rabbit colony has been
established and innoculation of these animals for antibody
production has been started° An epidemic of "snuffles;'
8
Figure 2o
Labyrinthectomized fish swimming inverted
in response to photostimulus from below.
Note control fish swimming normally.
9
a lagomorphic adenovirus, caused the loss of the original colony
but it appears that the second group is healthy.
We are currently preparing a paper for submission to
the Journal of Biological Chemistry describing this synthesis.
In addition, a note will be submitted to Copeia which briefly
describes the synthesis of this posterior pituitary hormone and
the significance of this work in providing material for studies
in teleost endocrinology.
3.3 Plans for the Coming Period
(a) Antibody production will be continued by innocu-
lating rabbits with purified synthetic arginine-vasotocin.
(b) Antibody presence in rabbit serum will be
determined.
(c) Antibody purification and conjugation with
Rhodamine B-200 will be attempted.
(d) Fish orientation studies will continue, attempt-
ing the use of rheotropic cues to augment the visual cue thus
hastening the development of stabilized inverted swimming
behaviors.
(e) Develop experimental plans for future phases of
this research effort.
i0
ACCESSION CUTOFF REPORT
ISSUENO. (CSTAR) I0
t
ISSUE ASSIGNHENT CUTOFF DATE 4/28/69
ACCESSIONING CUTOFF DATE 511169
4OO
TITLES ASSIGNED
TITLES TRANSFERRED (Includes pre-accession kills, rejects, & reassignments) 0
ACCESSION NUMBERS USED X69-14201 THROUGH X69-14600
0
ACCESSIONSNOT USED(At cutoff)
TOTAL ACCESSIONS (Including kills) 400
cc: E.E. Baker
W.L. Brown
T.T. Chapman
F.N. Cross
P.F. Eckert
J.W. Everett
B. Gilchrist
M.B. Hanes
M.G. Harris
A,P. Heizer
R.I. Hong
M.G. James
D. Kuny
H.A. Reger
C.W. Rusteburg
L.M. Schreiber
D.D. Thomas
J.J, Wal [i
W.E. White !


[Develop and Evaluate the Performance of Single and Multi-Specimen Respirometers.] Technical Report, 4 Nov. 1968 - 4 Feb. 1969

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