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PARTIAL CALLOSOTOMY AND LEFT-RIGHT RESPONSE
DIFFERENTIATION IN THE RAT: SEPARATE ANTERIOR AND POSTERIOR FACILITATORY EFFECTS
Michael Noonan and Seymour Axelrod
Canisius College and The State University of
New York at Buffalo
As published in Behavioral Neuroscience,
1992, 106, 433-436.
ACKNOWLEDGMENTS: This work was supported in part by a
grant to Canisius College from the Charles A. Dana Foundation, and by a Faculty
Research Fellowship to MN from Canisius College. The authors thank and praise
Jill Krakowiak, Debra Lipczynski, Michelle Penque and Julie Renda, who served as
research assistants. Sandra Thamer provided expert histological processing.
Direct inquiries and requests for reprints to Michael Noonan, Department of
Psychology, Canisius College, Buffalo, NY 14208.
ABSTRACT: Earlier work demonstrated that complete section
of the corpus callosum in the rat reduced the number of trials required to
acquire a left-right response differentiation (LRRD). We asked here whether the
facilitatory effect on LRRD could be produced by section of an anatomical
subdivision of the callosum. Rats with sections of the anterior or posterior
corpus callosum mastered the LRRD task faster than sham operates, but more
slowly than rats with total callosal section; section of the middle portion of
the callosum had no such effect. The partial facilitatory effects of anterior
and posterior callosotomy appear to be independent, and suggest that the
callosal intermixing of lateral information, which contributes to left-right
confusion, occurs at both the sensory and motor levels of processing.
INTRODUCTION
Distinguishing between left and right proves particularly
difficult for most animals (Corballis & Beale, 1976), and there is evidence from
experiments testing the effects of commissurotomy on left-right mirror-image
stimulus discrimination which suggests that the interhemispheric commissural
connections, by allowing lateralized signals to intermix, contribute to such
difficulty (Bykov & Speranski, 1924; Beale, Williams, Webster & Corballis, 1972;
Achim and Corballis, 1977).
Consistent with these lines of evidence is our recent
demonstration (Noonan & Axelrod, 1991 revision under review) that complete
corpus-callosum section in the rat reduced the number of trials required by rats
to acquire a left-right response differentiation (LRRD), while not affecting
performance in the same apparatus on tasks requiring brightness discrimination
or consistent unilateral responses. The question now arises whether the
difficulty in distinguishing left and right experienced by intact rats is
mediated by specifiable portions of the callosum. If it is, then section limited
to those portions should reproduce the effects of total callosotomy, and tracing
the projections of those portions might then yield insight into the neural
processes underlying the difficulty. Therefore, in a replication and extension
of our prior work, we conducted an investigation to ascertain whether
facilitatory effects on LRRD would result from selective sectioning of
anatomical subdivisions of the callosum.
PROCEDURES
Subjects. Adult male and female hooded (Long-Evans)
rats were maintained on an alternating 12:12 white:red light cycle, with all
testing occurring during the red phase. Room temperature was kept at 26°C. The
rats were randomly assigned to one of five surgical treatments: anterior
corpus-callosum section [ANTSECT], middle corpus-callosum section [MIDSECT],
posterior corpus-callosum section [POSTSECT], total corpus-callosum section [TOTSECT],
or sham surgery [SHAMSECT].
Surgery. Our callosotomy procedure has been
described in detail elsewhere (Noonan & Axelrod, 1991 revision under review).
Briefly, the scalp of the anesthetized rat was incised and an area of bone on
one side was thinned to allow a specially designed knife to be maneuvered under
the dura through a lateralized puncture wound in the adherent osteo-dural tissue
and, through a sequence of pivoting movements, into the longitudinal fissure
below the intact superior sagittal sinus. Movement of the knife in the mid-sagittal
plane then caused its sharpened leading edge to transect all or part of the
corpus callosum (and dorsal hippocampal commissure).
For the ANTSECT group, the knife was pivoted into the
longitudinal fissure 2 mm anterior to bregma, with the excursion of the cutting
edge extending back to bregma. For MIDSECT, the knife was pivoted into the
longitudinal fissure (and corpus callosum) just under bregma, making a 2 mm
excursion posteriorly from that point. For POSTSECT, the knife was pivoted at a
point 2 mm posterior to bregma, making a posterior excursion from that point
back to lambda. For TOTSECT, the knife was pivoted 2 mm in front of bregma,
making a posterior excursion to lambda. The SHAMSECT procedure mimicked the
TOTSECT procedure, but used a probe with a shortened leading edge that could
traverse the longitudinal fissure without cutting callosal tissue.
A minimum of six weeks postsurgical recovery was provided
before beginning behavioral testing, and in the week preceding testing each rat
was handled twice daily in order to habituate it to human contact.
Water Maze. We employed two water-mazes, which were
T-shaped plexiglass tanks, 46 cm deep. Water entered continuously (6.3 liter/min
at 24° C) at the floor of the starting box, and flowed out at the floor at the
end of both arms, being maintained at a depth of 25 cm. Each maze arm was 15 cm
wide and extended for 30 cm laterally. The arms then turned 90° back, so that an
escape ramp, extending down into the water at the end of the appropriate arm,
was out of sight to a rat at the choice point. The reinforcer was escape from
the water, accomplished by climbing up the ramp.
The walls of the maze were white and could be
back-illuminated. When not back-illuminated, the walls as viewed from within the
maze appeared to the human observer as uniform and dark. When back-illuminated,
alternating dark and light 3 cm-wide vertical stripes appeared.
LRRD Test. When the maze walls were illuminated,
the escape ramp was placed in the right arm, and when the walls were
unilluminated, the escape ramp was in the left arm. A "dummy" ramp, which
extended down only to 15 cm above the water surface and could therefore not be
used for escape, was always placed in the opposite arm so that the escape-ramp
location could not be determined by a view from outside the tank. Pseudorandomly
sequenced trials (maze-illuminated/maze-unilluminated) were presented at 4-min
inter-trial intervals. On each trial the rat was placed in the starting box and
allowed to swim until it found the ramp; it was then returned to its home cage
until the next trial. Each rat was tested for 25 trials per day until it reached
the criterion of 10 successive correct first turns at the choice point (or for a
maximum of five successive days). The number of trials taken to reach this
criterion served as the index of difficulty in making the left-right response
differentiation (cf. Noonan & Axelrod, 1989; 1990; 1991 revision under review).
All animal handling and data collection were carried out
by observers blind to the animals' surgical-group assignment, and recorded on
videotapes which were then reviewed by a second blind observer. In this and
related projects, we have also conducted a number of checks to confirm that our
subjects' scores were not functions of confounding variables: when testing was
continued beyond criterion, but by different handlers/observers, in different
rooms, and in different mazes oriented differently with respect to compass
heading, the subjects continued to demonstrate reliable LRRD.
Histology. Following behavioral testing, the rats
were perfused intracardially with saline followed by formalin. Serial 8-micron
coronal sections were made, with every 20th slice stained with cresyl violet.
Thirty rats were prepared in each of the five surgical
groups. LRRD data from two rats were spoiled by experimenter error during
testing, and were discarded. The LRRD data from nine additional rats were
subsequently excluded (without reference to LRRD scores) because the lesions
revealed by postmortem examination did not conform to those appropriate to any
of the intended subdivisions. The final group N's were: ANTSECT, 28; MIDSECT,
27; POSTSECT, 29; TOTSECT, 27; SHAMSECT, 28. Fifteen rats (2 ANTSECT, 6 MIDSECT,
2 POSTSECT, 0 TOTSECT, 5 SHAMSECT) failed to reach criterion after completing
the five days of testing (125 trials); they were assigned trials-to-criterion
LRRD scores equivalent to the lowest score possible given their ending
performance. As examples, if the response on the 125th trial was the last of 5
consecutive correct responses, the rat was given a score of 130. If the 125th
trial was incorrect, the rat was given a score of 135.
RESULTS
For each brain, we determined the number of stained
sections in which callosal fibers would have crossed the midline had the brain
remained intact, but in which the fibers were severed. For the TOTSECT subjects
this value averaged 15.3, representing 94% of the sections in which midline
callosal fibers either appeared, or would have appeared. For ANTSECT, MIDSECT,
POSTSECT and SHAMSECT these values were 5.3 (35%), 6.2 (40%), 8.0 (54%), and 0.0
(0%) respectively. Sections of representative brains in each group are presented
in Figure 1.
Examination of the brain sections also showed that most
animals suffered a degree of incidental surgical damage to the midline cortex,
and sometimes also to the septum, fimbria, hippocampus, thalamus, or tectum,
comparable to that reported by Noonan and Axelrod (1991 revision under review).
Representative examples are provided in Figure 1. Analyses parallel to those
presented in the earlier report (ANOVAs comparing the LRRD scores of those with
and those without such damage, or Pearson correlations testing the relation
between the extent of such damage and LRRD scores, within each group) showed, as
did that report, that LRRD scores were not related to the presence or extent of
damage to any of these structures.
The mean number of trials to reach criterion on the
left-right response differentiation task for each group is shown in Figure 2. A
one-way ANOVA showed the five means to differ reliably, F (4, 134) =
4.21, p = .003. Application of the Cochran C, the Bartlett-Box
F, and the Maximum/Minimum Variance tests all indicated that the
within-groups variances were not significantly different from homogeneous.
Subsequent one-tail t tests were applied, using the within-groups mean
square from the ANOVA as the estimate of error variance, testing for each
lesion-group mean the hypothesis that it was lower than the SHAMSECT mean. As in
our earlier study, the TOTSECT group required significantly fewer trials than
the shams, t (134) = 3.51, p = .0003. Both the ANTSECT group (t
[134] = 1.90, p = .030) and the POSTSECT group (t [134] = 2.38,
p = .011) likewise had significantly lower scores than the shams; the
difference between MIDSECT and SHAMSECT, although in the hypothesized direction,
was negligible (t [134] = 0.33).
The LRRD scores in this experiment were on average higher
than those found in our earlier study (Noonan & Axelrod, 1991 revision under
review). For example, the mean scores of the complete-callosotomy and sham
groups were 62.1 and 89.4 respectively in the present study, compared with 49.3
and 65.4 respectively in the earlier study. There were at least two differences
between the samples in the two studies which might account for the differences
in score. First, the rats used in the previous work were commercially bred and
purchased at about 35 days of age, whereas the rats in the present experiment
were bred in our laboratory. Second, the rats in the earlier study were tested
at 150-170 days of age, as contrasted with 102-133 days in the present study.
Whatever may be the source of the difference between the studies in the general
level of scores, it will be noted that in both the improvement of the
complete-callosotomy animals over their respective sham controls was significant
and of approximately the same order (30.5% improvement in the present
experiment, 24.6% in the previous work).
DISCUSSION
The decision to subdivide the corpus callosum into three
antero-posterior portions was a reasonable, if arbitrary, first step in our
effort to narrow down the facilitatory effect of callosotomy on LRRD to a
specifiable callosal subdivision. These callosal subdivisions were not however
equal in terms of the proportion of callosal tissue included; the anterior and
posterior portions of the callosum (the genu and splenium) are thicker than the
trunk. In light of this, the negligible and non-significant improvement of the
MIDSECT group relative to the SHAMs might have been due to the relatively small
proportion of the total callosum having been sectioned in this group. It might
be hypothesized that the facilitatory effect on LRRD performance of callosotomy
is simply a function of the proportion of callosal fibers sectioned,
irrespective of the location of the cut fibers.
A direct test of such a "mass-action" hypothesis would
require deliberate variation of lesion size at a number of callosal loci.
However, the following post-hoc analysis of the present data would appear to
make such an account unlikely. We calculated the callosal midline area present
in those sections of each sham brain corresponding to those which had been
severed in each callosotomy subject, and we then assigned the mean of these
calculated areas to each callosotomy subject. For example, if an ANTSECT brain
showed the corpus callosum to have been severed in the first 5 of the 15
sections in which it either crossed or would have crossed the midline, we
calculated for each of the 28 sham brains the sum of the midsagittal thicknesses
of the corpus callosum in each of the first one-third of the sections in which
the corpus callosum crossed the midline (where this procedure did not yield an
integral number of sections, the sum was adjusted by linear interpolation). This
sum, indexing the midline callosal area in the sham brain corresponding to the
callosal portion which had been severed in the sectioned brain, was then divided
by the sum of the midline callosal thicknesses from all sections, indexing the
sham brain's total callosal area. The 28 proportions thus produced were averaged
to yield an estimate of the proportion of the callosum severed in the sectioned
brain. (The mean values of these estimates were 44.8% for the ANTSECT group,
29.7% for MIDSECT, 44.1% for POSTSECT, and 92.4% for TOTSECT.)
LRRD performance turned out not to be a function of these
estimates: the Pearson product-moment correlations between LRRD score and
proportion of callosum cut across all the partial callosotomy subjects, and
within each partial-callosotomy group, were non-significant and variable in
sign. It appears then that the facilitatory effect of callosal section does not
depend upon the location-independent magnitude of the section. Rather,
facilitation was produced by sectioning of the anterior and posterior callosal
fibers specifically.
That the ANTSECT and POSTSECT groups both had LRRD scores
intermediate between those of the TOTSECT and SHAMSECT groups suggests that the
facilitative effect on LRRD of full callosotomy derives from two separate
contributions, one associated with severing the anterior callosal fibers, the
other with severing the posterior fibers. It is possible that the callosal
intermixing of left- and right-specific information contributing to the
difficulty of distinguishing left and right occurs at both the motor (anterior)
and sensory (posterior) levels of processing. Whatever their nature, the
contributions appear to be additive: the magnitude of the facilitatory effect of
complete callosotomy (expressed as the difference between the TOTSECT and
SHAMSECT means, 27.3 trials) is close to the sum (32.8) of the facilitatory
effects of anterior sectioning (14.6) and posterior sectioning (18.2). It now
remains to be determined whether these partial contributions can be further
subdivided, and whether our ability to localize them anatomically can be further
refined so that the origins and terminations of the implicated callosal fibers
can be identified.
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