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Neuronal isolation by combined midline and transverse section of the cat brain
Life Sciences Vol . 5, pp . 12~~-1282, 1966 . Printed in Great Britain.
Pergamon Press Ltd.
NEURONAL ISOLATION BY COMBINED MIDLINE AND TRANSVERSE SECTION OF THE CAT BRAINI Theodore J . Voneida Department of Anatomy, School of Medicine Western Reserve University, Cleveland, Ohio 44106
(Received 9 May 1966 ; in final form 26 May 1966) In vivo isolation of neural tissue has been accomplished by several techniques, dating back to the decerebrate preparations of C . S . Sherrington (1) .
These
have provided neurophysiologists with a means of examining large segments of the central nervous system in various states of relative isolation .
A major
problem with these preparations is that animals with midbrain or bulbospinal transections are extremely difficult to maintain .
They rarely survive for
more than a few weeks, during which time they exhibit decerebrate rigidity and loss of temperature regulation .
More recently, White et al (2, 3) have re-
ported maintenance of electrocortical activity for several hours to 6 days during anatomic isolation of the monkey brain and the dog brain when circulation was provided by compatible donors .
Gilboe et al (4) have achieved
similar results with the isolated dog brain, the viability of which was maintained for several hours by a mechanical perfusion technique .
Kellaway, Gol
and Proler (5) have also described a technique by which it is possible to completely isolate the cortex of one cerebral hemisphere from the remainder of the brain while preserving its blood supply and drainage . The above methods provide numerous possibilities for pharmacological and electrophysiological examination of central nervous structures, but at the same time they impose severe limitations on studies which require long-term observa tion .
A technique is described below by which fore- and midbrain structures
1 This investigation was supported by a research grant (Np-I-07051) from the United States Public Health Service .
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can be isolated as a unit from the rest of the brain without causing the severe incapacitation resulting from complete transection of the brain stem . Methods This procedure involves a unilateral brain stem transection plus . an extension of the split brain preparation of Myers and Sperry (6) to include not only the optic chiasm, forebrain and habenular commissures, but also the massa intermedia, posterior, and tectal commissures, as well as the midbrain tegmentum (Fig . 1) .
The midline section has been carried out successfully in
numerous animals as part of a series of studies related to central mechanisms in visually guided behavior (7, 8, 9, 10) .
It is possible to section the above
structures simultaneously, thus severing all connections between the two brain halves, as well as eliminating the crossed optic fibers . The next stage of this procedure involves unilateral transection of the brain stem at precollicular, intercollicular or postcollicular levels, thus isolating forebrain or forebrain plus midbrain structures of one hemisphere from neural communication with the remainder of the brain .
Visual and ol-
factory input can be eliminated by appropriate tract section, although this was not done in the preparations described below . Results and Discussion All animals recover from . the midline section exclusive ôf tegmentum within 1 to 2 days, and except for the chronic pupillary dilation (11) and a transient 'high stepping' gait, they are difficult to differentiate from normal .
Ex
tension of the midline section ventrally to include the mesencephalic tegmentum results in a severe and extended loss of the ability to respond to visual cues (10), but otherwise the subjects are capable of maintaining themselves and of carrying out normal activities, such as walking, grooming and eating . Midline section plus unilateral intercollicular transection has been attempted in 3 animals to date .
One died as a result of hemmorhage following
the transverse section, but the other 2 recovered and were observed under various conditions for 6 and 10 months, respectively, when they were perfused
Vol .
5,
No .
14
127 9
NEURONAL ISOLATION
Fig . 1 Representative transverse sections (stained by the Weil method) showing midline cut of optic chiasm, fore- and midbrain commissures, and the midbrain tegmentum . and the brains removed for histologic examination .
The immediate postoperative
period (4 - 5 days) is characterized in these animals by a severe twisting of the body and head toward the side of the transverse lesion . attempts to walk were made after about one week .
Uncoordinated
They are charactérized by a
pronounced weakness of the limbs contralateral to the section, and forced circling toward the side of the lesion .
These symptoms gradually diminish,
1280
NEURONAL ISOLATION
Vol .
5,
No .
14
and within 4 - 5 weeks the animals are capable of walking with little difficulty . It is necessary to assist them in eating during the first 5 - 10 postoperative days, after which they are capable of maintaining themselves independently . A measure of the degree of neural isolation may be gained by comparing electrocortical activity as recorded from subdural electrodes implanted bilaterally (2 in the rostral third of the lateral gyros, 2 in the anterior supra sylvian gyros, and one in the caudal third of the lateral gyros) in normal, midline sectioned and midline plus transverse sectioned (intercollicular level) animals (Fig . 2) .
It is evident that interhemispheric cortical activity in
normal and midline sectioned animals is coordinated (Figs . 2A and B) .
Inter-
hemispheric coordination is seen only intermittently, however, following the isolation procedure described above .
The isolated segment often exhibits sleep-
like patterns (Fig . 2C, lower trace), while the non-isolated segment is in a wakeful state (Fig . 2C, upper trace) .
These periods of uncoordinated activity
alternate with periods during which both hemispheres exhibit somewhat similar patterns (Fig . 2D) .
It is extremely rare, however, to find the degree of
interhemispheric symmetry which typifies normal and midline sectioned animals . The above records, which were made 10 months after surgery, show no greater signs of coordinated activity than do records made during the first 2 weeks following isolation . Several studies have been reported in which asymmetric activity has been demonstrated between the hemispheres for short periods of time under various experimental conditions .
Knott, Ingram and Chiles (12) produced a transient
slowing of the electrical activity of the ipsilateral hemisphere following chronic lesions limited to one side of the midbrain tegmentum . was found to disappear within a few days .
This asymmetry
Similar results were reported by
Cordeau and ~fancia (13) following unilateral chronic lesions of the midbrain, in which interhemispheric asymmetry occurred initially, but disappeared completeiy within 5 to 8 days postoperatively .
It is conceivable that the early
return of symmetry reported in the above studies could be attributed to the
Vol . 5, No . 14
NEURONAL ISOLATION
1281
Fig . 2 Electrocortical activity as recorded between subdurally implanted electrodes in the lateral and suprasylvian gyri of the left (upper trace) and right (lower trace) hemispheres . (A) Normal animal, 1 month following elec trode implanta (B) Midline sectioned animal (optic chiasm, massa intermedia, corpus callosum, anterior, posterior, habenular, hippocampal, and tectal commissures) 13 months postoperatively, (C,D) Animal with midline section (gs in B) plus unilateral transverse section of brain stem at intercollicular level, 10 months postoperatively . presence of commissural connections, all of which remained intact .
This
possibility is reinforced by the present, results, in which a greatly prolonged period of interhemispheric asymmetry is observed following unilateral brain stem transection plus complete commissurotomy .
There are periods, however,
during which an electrical symmetry does exist between the isolated and nonisolated segments (Fig . 2D) .
The basis bf the symmetry is difficult to ex-
plain in the absence of neuronal connections, but the possibility exists, as has been suggested by Ingvar (14) and by Graham Brown (15), that humoral or circulatory factors may play a role in regulating electroçortical activity . Summary The hemi-decerebrate preparation described above provides a means by which
1282
NEURONAL ISOLATION
Vol. 5, No . 14
long term studies can be made on relatively large segments of the brain which have a normal vascular supply, but which are neuronally isolated from the remainder of the central nervous system .
Complete isolation can be achieved
by eliminating inptit from the first two cranial nerves by appropriate tract sections .
The absence of commissural connections is cited as a possible ex-
planation for the prolonged periods of asymmétric electrocortical activity between isolated and non-isolated segments .
It is suggested that the brief
periods of symmetriç activity may be due to humoral or circulatory factors . Acknowledgment-I wish to thank Mr . Ralph Morgan for his assistance in this work . References 1.
C .S . SHERRINGfON, J . Physiol . London 22, 319 (1898) .
2.
R .J . iVHITE, M .S . ALBIN and J . VERDURA, Science 141, 1060 (1963) .
3.
, G .E . LOCKE and E . DAVIDSON, Ibid . 150, 779 (1965) .
4.
D .D . GILBOE, W .W . COTANCK and M .B . GLOVER, Nature 206, 94 (1965) .
5.
P . KELLAWAY, A GOL and M . PROLER, Exp . Neurol . 14, 281 (1966) .
6.
R . E . MYERS and R .W . SPERRY, Anat . Rec .
7.
T .J . V~IEIDA and R .W . SPERRY, Anat . Rec . 139 , 283 (1961) .
8.
T .J . VOiNEIDA, Ibid . 142, 287 (1962) .
115, 351 (1953) .
9.
, Exp . Neurol . 8, 493 (1963) .
10 .
, Anat . Rec . 151, 429 (1965) .
11 .
T .H . CAULFIELD, J .S . ROBINSOri and T .J . VONEIDA, Amer . Psychol . 19, 490 (1964) .
12 .
J .R . KNOTT, W .R . INGRAM and W .D . CHILES, Arch . Neurol . P sychiat . Chicago 73, 203 (1955) .
13 .
J .P . CORDEAU and M . MANCIA, Arch . ital . Biol . 96, 374 (1958) .
14 .
D .H . INGVAR, Acta Physiol . Scand . 33, 169 (1955) .
15 .
T . GRAHAM BROWN, J . Ph~siol . - London 48, 18 (1914) .
Pergamon Press Ltd.
NEURONAL ISOLATION BY COMBINED MIDLINE AND TRANSVERSE SECTION OF THE CAT BRAINI Theodore J . Voneida Department of Anatomy, School of Medicine Western Reserve University, Cleveland, Ohio 44106
(Received 9 May 1966 ; in final form 26 May 1966) In vivo isolation of neural tissue has been accomplished by several techniques, dating back to the decerebrate preparations of C . S . Sherrington (1) .
These
have provided neurophysiologists with a means of examining large segments of the central nervous system in various states of relative isolation .
A major
problem with these preparations is that animals with midbrain or bulbospinal transections are extremely difficult to maintain .
They rarely survive for
more than a few weeks, during which time they exhibit decerebrate rigidity and loss of temperature regulation .
More recently, White et al (2, 3) have re-
ported maintenance of electrocortical activity for several hours to 6 days during anatomic isolation of the monkey brain and the dog brain when circulation was provided by compatible donors .
Gilboe et al (4) have achieved
similar results with the isolated dog brain, the viability of which was maintained for several hours by a mechanical perfusion technique .
Kellaway, Gol
and Proler (5) have also described a technique by which it is possible to completely isolate the cortex of one cerebral hemisphere from the remainder of the brain while preserving its blood supply and drainage . The above methods provide numerous possibilities for pharmacological and electrophysiological examination of central nervous structures, but at the same time they impose severe limitations on studies which require long-term observa tion .
A technique is described below by which fore- and midbrain structures
1 This investigation was supported by a research grant (Np-I-07051) from the United States Public Health Service .
128
NEURONAL ISOLATION
Vol .
5,
No .
14
can be isolated as a unit from the rest of the brain without causing the severe incapacitation resulting from complete transection of the brain stem . Methods This procedure involves a unilateral brain stem transection plus . an extension of the split brain preparation of Myers and Sperry (6) to include not only the optic chiasm, forebrain and habenular commissures, but also the massa intermedia, posterior, and tectal commissures, as well as the midbrain tegmentum (Fig . 1) .
The midline section has been carried out successfully in
numerous animals as part of a series of studies related to central mechanisms in visually guided behavior (7, 8, 9, 10) .
It is possible to section the above
structures simultaneously, thus severing all connections between the two brain halves, as well as eliminating the crossed optic fibers . The next stage of this procedure involves unilateral transection of the brain stem at precollicular, intercollicular or postcollicular levels, thus isolating forebrain or forebrain plus midbrain structures of one hemisphere from neural communication with the remainder of the brain .
Visual and ol-
factory input can be eliminated by appropriate tract section, although this was not done in the preparations described below . Results and Discussion All animals recover from . the midline section exclusive ôf tegmentum within 1 to 2 days, and except for the chronic pupillary dilation (11) and a transient 'high stepping' gait, they are difficult to differentiate from normal .
Ex
tension of the midline section ventrally to include the mesencephalic tegmentum results in a severe and extended loss of the ability to respond to visual cues (10), but otherwise the subjects are capable of maintaining themselves and of carrying out normal activities, such as walking, grooming and eating . Midline section plus unilateral intercollicular transection has been attempted in 3 animals to date .
One died as a result of hemmorhage following
the transverse section, but the other 2 recovered and were observed under various conditions for 6 and 10 months, respectively, when they were perfused
Vol .
5,
No .
14
127 9
NEURONAL ISOLATION
Fig . 1 Representative transverse sections (stained by the Weil method) showing midline cut of optic chiasm, fore- and midbrain commissures, and the midbrain tegmentum . and the brains removed for histologic examination .
The immediate postoperative
period (4 - 5 days) is characterized in these animals by a severe twisting of the body and head toward the side of the transverse lesion . attempts to walk were made after about one week .
Uncoordinated
They are charactérized by a
pronounced weakness of the limbs contralateral to the section, and forced circling toward the side of the lesion .
These symptoms gradually diminish,
1280
NEURONAL ISOLATION
Vol .
5,
No .
14
and within 4 - 5 weeks the animals are capable of walking with little difficulty . It is necessary to assist them in eating during the first 5 - 10 postoperative days, after which they are capable of maintaining themselves independently . A measure of the degree of neural isolation may be gained by comparing electrocortical activity as recorded from subdural electrodes implanted bilaterally (2 in the rostral third of the lateral gyros, 2 in the anterior supra sylvian gyros, and one in the caudal third of the lateral gyros) in normal, midline sectioned and midline plus transverse sectioned (intercollicular level) animals (Fig . 2) .
It is evident that interhemispheric cortical activity in
normal and midline sectioned animals is coordinated (Figs . 2A and B) .
Inter-
hemispheric coordination is seen only intermittently, however, following the isolation procedure described above .
The isolated segment often exhibits sleep-
like patterns (Fig . 2C, lower trace), while the non-isolated segment is in a wakeful state (Fig . 2C, upper trace) .
These periods of uncoordinated activity
alternate with periods during which both hemispheres exhibit somewhat similar patterns (Fig . 2D) .
It is extremely rare, however, to find the degree of
interhemispheric symmetry which typifies normal and midline sectioned animals . The above records, which were made 10 months after surgery, show no greater signs of coordinated activity than do records made during the first 2 weeks following isolation . Several studies have been reported in which asymmetric activity has been demonstrated between the hemispheres for short periods of time under various experimental conditions .
Knott, Ingram and Chiles (12) produced a transient
slowing of the electrical activity of the ipsilateral hemisphere following chronic lesions limited to one side of the midbrain tegmentum . was found to disappear within a few days .
This asymmetry
Similar results were reported by
Cordeau and ~fancia (13) following unilateral chronic lesions of the midbrain, in which interhemispheric asymmetry occurred initially, but disappeared completeiy within 5 to 8 days postoperatively .
It is conceivable that the early
return of symmetry reported in the above studies could be attributed to the
Vol . 5, No . 14
NEURONAL ISOLATION
1281
Fig . 2 Electrocortical activity as recorded between subdurally implanted electrodes in the lateral and suprasylvian gyri of the left (upper trace) and right (lower trace) hemispheres . (A) Normal animal, 1 month following elec trode implanta (B) Midline sectioned animal (optic chiasm, massa intermedia, corpus callosum, anterior, posterior, habenular, hippocampal, and tectal commissures) 13 months postoperatively, (C,D) Animal with midline section (gs in B) plus unilateral transverse section of brain stem at intercollicular level, 10 months postoperatively . presence of commissural connections, all of which remained intact .
This
possibility is reinforced by the present, results, in which a greatly prolonged period of interhemispheric asymmetry is observed following unilateral brain stem transection plus complete commissurotomy .
There are periods, however,
during which an electrical symmetry does exist between the isolated and nonisolated segments (Fig . 2D) .
The basis bf the symmetry is difficult to ex-
plain in the absence of neuronal connections, but the possibility exists, as has been suggested by Ingvar (14) and by Graham Brown (15), that humoral or circulatory factors may play a role in regulating electroçortical activity . Summary The hemi-decerebrate preparation described above provides a means by which
1282
NEURONAL ISOLATION
Vol. 5, No . 14
long term studies can be made on relatively large segments of the brain which have a normal vascular supply, but which are neuronally isolated from the remainder of the central nervous system .
Complete isolation can be achieved
by eliminating inptit from the first two cranial nerves by appropriate tract sections .
The absence of commissural connections is cited as a possible ex-
planation for the prolonged periods of asymmétric electrocortical activity between isolated and non-isolated segments .
It is suggested that the brief
periods of symmetriç activity may be due to humoral or circulatory factors . Acknowledgment-I wish to thank Mr . Ralph Morgan for his assistance in this work . References 1.
C .S . SHERRINGfON, J . Physiol . London 22, 319 (1898) .
2.
R .J . iVHITE, M .S . ALBIN and J . VERDURA, Science 141, 1060 (1963) .
3.
, G .E . LOCKE and E . DAVIDSON, Ibid . 150, 779 (1965) .
4.
D .D . GILBOE, W .W . COTANCK and M .B . GLOVER, Nature 206, 94 (1965) .
5.
P . KELLAWAY, A GOL and M . PROLER, Exp . Neurol . 14, 281 (1966) .
6.
R . E . MYERS and R .W . SPERRY, Anat . Rec .
7.
T .J . V~IEIDA and R .W . SPERRY, Anat . Rec . 139 , 283 (1961) .
8.
T .J . VOiNEIDA, Ibid . 142, 287 (1962) .
115, 351 (1953) .
9.
, Exp . Neurol . 8, 493 (1963) .
10 .
, Anat . Rec . 151, 429 (1965) .
11 .
T .H . CAULFIELD, J .S . ROBINSOri and T .J . VONEIDA, Amer . Psychol . 19, 490 (1964) .
12 .
J .R . KNOTT, W .R . INGRAM and W .D . CHILES, Arch . Neurol . P sychiat . Chicago 73, 203 (1955) .
13 .
J .P . CORDEAU and M . MANCIA, Arch . ital . Biol . 96, 374 (1958) .
14 .
D .H . INGVAR, Acta Physiol . Scand . 33, 169 (1955) .
15 .
T . GRAHAM BROWN, J . Ph~siol . - London 48, 18 (1914) .