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The orientation of mitral cell dendrites
EXPERIMENTAL
NEUROLOGY
The
14, 390-395
Orientation
(1966)
of Mitral G. M.
National
Institutes Received
Cell
Dendrites
SHEPHERDS
of Health,
September
Bethesda, 17,
Maryland
1965
Inhibition of mitral cells in the olfactory bulb of the rabbit appears to be mediated through their secondary dendrites in the external plexiform layer. These dendrites can be clearly seen in Nissl-stained paraffin sections when fixation of the bulb has been carried out with Bouin’s fluid. An anteroposterior bias in the orientation of the mitral secondary dendrites was found by comparing sections cut in different planes of the bulb. The results suggest that the inhibitory systems which control the mitral cells have a longitudinal orientation in the bulb of the rabbit. Introduction
and
Method
Microelectrode experiments on the rabbit’s olfactory bulb have indicated that a powerful inhibition of mitral cells is mediated through their secondary or basal dendrites (3, 7, 8). In view of this important functional role it is of interest that a predominantly longitudinal orientation of these dendrites in the bulb has been found, using a routine histological procedure. Young rabbits (less than 3 months old and 2 kg weight) were used to avoid the high incidence of atrophic rhinitis in older animals. They were deeply anesthetized with nembutal, and perfused by gravity through the beating heart with warm saline solution followed by Bouin’s fluid. A period of 3-4 hours passed before removal of the bulbs in order to avoid trauma in the immediate postmortem period, which is believed to be responsible for “dark neurons” (2). The bulbs were then fixed overnight in Bouin’s fluid, washed in several changes of 50% alcohol during the following day and transferred to 7Oc/( alcohol. Paraffin sections 1.5 p thick were cut in the sagittal, frontal or horizontal planes and stained with cresyl-violet acetate (Allied Chemical and Dye Corp.), in 0.25% solution. The same techniques were used in other bulbs which had been perfused and fixed in 1 Dr. Wade Marshall, Dr. Grant Rasmussen and Dr. Paul MacLean generously provided the facilities for this study. I am indebted to Dr. Jan Cammermeyer, Mr. George Creswell and Mr. John Lewis for technical advice and assistance, and to DrWilfrid Rall and Dr. Thomas Reese for valuable discussions. 390
MITRAL
10% formalin. method.
Alternate
391
DENDRITES
groups of sections were stained by a Weigert Results
and
Discussion
The cell bodies and nuclei of the different layers of the olfactory bulb were clearly stained, as expected with this Nissl-type stain. In addition, and unexpectedly, the dendritic processes in the external plexiform layer were also clearly seen (Fig. 1) . They were readily distinguished from small blood vessels. These dendrites could also be visualized in material fixed in Bouin’s fluid and stained by the Weigert method. Formalin-fixed material, on the other hand, even when deeply stained by cresyl-violet or the Weigert method, showed little of this detail. From the classical studies with Golgi’s method (6)) it is known that each mitral cell of the rabbit sends usually a single C‘primary” dendrite to a
GLOMERULI
tufted
cell
body
blood
vess el
EXTERNAL PLEXIFORM
primal ‘y de ndrite
MITRAL
BODY radral
process
GRANULE FIG. 1. Sagittal section of rabbit’s olfactory bulb; dorsal region. Bouin’s fixation, paraffin embedding, cresyl-violet stain, 15-p section. Histological layers are indicated on the left; the bulb surface is above. Magnification x 180.
392
SHEPHERD
glomerulus and several “secondary” dendrites into the external plexiform layer. The section in Fig. 1 was cut parallel to a mitral primary dendrite, and shows its entire extent from the cell body to the glomerulus. Such observations were frequent in the thick sections. The primary dendrite diminishes only slightly in diameter during its course toward the glomerulus, and is rarely branched in the rabbit. In Fig. 1 several other large dendrites, presumably primary because of their radial orientation, can be seen entering the glomerulus. The primary dendrites course through a dense matrix of smaller dendrites which run tangentially or horizontally. Deep in the plexiform Iayer these are mostly mitral secondary dendrites and their branches; near the glomeruli they arise mostly from the tufted cells which lie in that region. h-0 demarcation between these dendritic zones was apparent, however. The primary and secondary dendrites are structurally indistinguishable with this stain. This is consistent with the observation that a primary dendrite occasionally shares a common trunk with a secondary dendrite, as has also been seen with Golgi stains (6). Figure 2 illustrates an attempt to show the disposition of the secondary
FIG. 2. Sagittal section through the of the bulb. Anterior is above, posterior
mitral below.
body layer Cresyl-violet.
of
the midmedial Magnification x
region 450.
MITRAL
393
DENDRITES
dendrites as they extend horizontally from the mitral cell bodies. The section is parallel to and within the layer of mitral cell bodies, so that we are looking down on the layer from above, as it were. The dendritic trunks and branches form a tortuous meshwork running generally parallel to the plane of section; cross-cut dendrites are rare. These secondary dendrites are too tortuous and branched to be followed for any great distance but there appears to be an anteroposterior bias in the way the dendrites run, although it is difficult to characterize. More quantitative evidence for an anteroposterior bias in the orientation of the mitral secondary dendrites was obtained by comparing sections in different planes in the bulb. Figure 3 is from a frontal section, and shows
FIG.
bulbar
3. Frontal section, mid-anteroposterior in surface is above. Cresyl-violet. Magnification
bulb, dorsal X $50.
bulbar
region.
The
the external plexiform layer profusely pock-marked by small bodies. A few of these are clearly blood vessels; the rest are horizontally and tangentially running dendrites cut in cross section. In the deep part of the external plexiform layer, these are mostly mitral secondary dendrites. By comparison, in the longitudinal plane of Fig. I, the L’holes” in the section are few and many horizontal and tangential dendritic fragments can be seen running more or less in the plane of the section.
SHEPHERD
394
When the number of cross-cut dendrites per area of field was counted, the frontal sections through the deep half of the external plexiform layer consistently showed about three times the number seen in longitudinal sections. This difference was significant at the p < 0.01 level. The difference was seen when comparing the two planes of section in the same bulb and also when comparing the two planes of section in different bulbs (as in Figs. I and 3). While the density of cross-cut dendrites in frontal sections was more or less constant throughout the circumference of the bulb, the diameters tended to be smaller in the ventral than in the dorsal external plexiform layer. At the anterior pole of the bulb the three planes of sections showed no significant differences. Evidence has recently been presented that dendrodendritic synapses between mitral secondary dendrites and granule processes provide the pathway for the powerful recurrent inhibition of the mitral cells which has been demonstrated in microelectrode experiments (4). The mitral dendrites which mediate this inhibition have an anteroposterior orientation; they thus tend to parallel the mitral axons running deeper in the bulb, sending collaterals to the granule and external plexiform layers; they also parallel the numerous collaterals from the tufted cell axons running in an anteroposterior direction in the internal plexiform layer (5). One can thus conceive of the rabbit’s olfactory bulb as organized in longitudinal wedges, like the sections of an orange. The functional significance of this organization might be that it allows a spread of mitral inhibition in the anteroposterior direction, while restricting it in the mediolateral direction. Possibly this is a partial basis for Adrian’s (1) finding of an anteroposterior gradient in the intensities of the responses of mitral cells to certain odors. References 1. 2.
3.
4.
5.
D. 1953. Sensory messages and sensation. The response of the olfactory organ to different smells. Acta Physiol. Sand. 29: 5-14. CAMMERMEYER, J. 1961. The importance of avoiding “dark” neurons in experimental neuropathology. Acta Neuropathol. 1: 245-270. PHILLIPS, C. G., T. P. S. POWELL, and G. M. SHEPHERD. 1963. Responses of mitral cells to stimulation of the lateral olfactory tract in the rabbit, J. Physiol. London 166: 65-88. RALL, W., G. M. SHEPHERD, T. S. REESE, and M. W. BRIGHTMAN. 1966. Dendrodendritic synaptic pathway for inhibition in the olfactory bulb. Exptl. Neuuol. 14: 44-56. RAMON Y CAJAL, S. 1891. Origin y termination de las fibras nerviosas olfactorias. Gac. Sanit. Barcelona 3: 133-139; 174-181; 206-212.
ADRIAN,
E.
MITRAL
6.
7. 8.
DENDRITES
395
RAMON Y CAJAL, S. 1911. “Histologie du Systizme Nerveux de I’Homme et des Verthbrks,” 2. L. Azoulay [trans.] Maloine, Paris. SHEPHERD, G. M. 1963. Neuronal systems controlling mitral cell excitability. J. Physiol. London 166: 101-117. YAMAMOTO, C., T. YAMAMOTO, and K. IWAMA. 1963. The inhibitory systems in the olfactory bulb studied by intracellular recording. J. Neurophysiol. 26: 403-415.
NEUROLOGY
The
14, 390-395
Orientation
(1966)
of Mitral G. M.
National
Institutes Received
Cell
Dendrites
SHEPHERDS
of Health,
September
Bethesda, 17,
Maryland
1965
Inhibition of mitral cells in the olfactory bulb of the rabbit appears to be mediated through their secondary dendrites in the external plexiform layer. These dendrites can be clearly seen in Nissl-stained paraffin sections when fixation of the bulb has been carried out with Bouin’s fluid. An anteroposterior bias in the orientation of the mitral secondary dendrites was found by comparing sections cut in different planes of the bulb. The results suggest that the inhibitory systems which control the mitral cells have a longitudinal orientation in the bulb of the rabbit. Introduction
and
Method
Microelectrode experiments on the rabbit’s olfactory bulb have indicated that a powerful inhibition of mitral cells is mediated through their secondary or basal dendrites (3, 7, 8). In view of this important functional role it is of interest that a predominantly longitudinal orientation of these dendrites in the bulb has been found, using a routine histological procedure. Young rabbits (less than 3 months old and 2 kg weight) were used to avoid the high incidence of atrophic rhinitis in older animals. They were deeply anesthetized with nembutal, and perfused by gravity through the beating heart with warm saline solution followed by Bouin’s fluid. A period of 3-4 hours passed before removal of the bulbs in order to avoid trauma in the immediate postmortem period, which is believed to be responsible for “dark neurons” (2). The bulbs were then fixed overnight in Bouin’s fluid, washed in several changes of 50% alcohol during the following day and transferred to 7Oc/( alcohol. Paraffin sections 1.5 p thick were cut in the sagittal, frontal or horizontal planes and stained with cresyl-violet acetate (Allied Chemical and Dye Corp.), in 0.25% solution. The same techniques were used in other bulbs which had been perfused and fixed in 1 Dr. Wade Marshall, Dr. Grant Rasmussen and Dr. Paul MacLean generously provided the facilities for this study. I am indebted to Dr. Jan Cammermeyer, Mr. George Creswell and Mr. John Lewis for technical advice and assistance, and to DrWilfrid Rall and Dr. Thomas Reese for valuable discussions. 390
MITRAL
10% formalin. method.
Alternate
391
DENDRITES
groups of sections were stained by a Weigert Results
and
Discussion
The cell bodies and nuclei of the different layers of the olfactory bulb were clearly stained, as expected with this Nissl-type stain. In addition, and unexpectedly, the dendritic processes in the external plexiform layer were also clearly seen (Fig. 1) . They were readily distinguished from small blood vessels. These dendrites could also be visualized in material fixed in Bouin’s fluid and stained by the Weigert method. Formalin-fixed material, on the other hand, even when deeply stained by cresyl-violet or the Weigert method, showed little of this detail. From the classical studies with Golgi’s method (6)) it is known that each mitral cell of the rabbit sends usually a single C‘primary” dendrite to a
GLOMERULI
tufted
cell
body
blood
vess el
EXTERNAL PLEXIFORM
primal ‘y de ndrite
MITRAL
BODY radral
process
GRANULE FIG. 1. Sagittal section of rabbit’s olfactory bulb; dorsal region. Bouin’s fixation, paraffin embedding, cresyl-violet stain, 15-p section. Histological layers are indicated on the left; the bulb surface is above. Magnification x 180.
392
SHEPHERD
glomerulus and several “secondary” dendrites into the external plexiform layer. The section in Fig. 1 was cut parallel to a mitral primary dendrite, and shows its entire extent from the cell body to the glomerulus. Such observations were frequent in the thick sections. The primary dendrite diminishes only slightly in diameter during its course toward the glomerulus, and is rarely branched in the rabbit. In Fig. 1 several other large dendrites, presumably primary because of their radial orientation, can be seen entering the glomerulus. The primary dendrites course through a dense matrix of smaller dendrites which run tangentially or horizontally. Deep in the plexiform Iayer these are mostly mitral secondary dendrites and their branches; near the glomeruli they arise mostly from the tufted cells which lie in that region. h-0 demarcation between these dendritic zones was apparent, however. The primary and secondary dendrites are structurally indistinguishable with this stain. This is consistent with the observation that a primary dendrite occasionally shares a common trunk with a secondary dendrite, as has also been seen with Golgi stains (6). Figure 2 illustrates an attempt to show the disposition of the secondary
FIG. 2. Sagittal section through the of the bulb. Anterior is above, posterior
mitral below.
body layer Cresyl-violet.
of
the midmedial Magnification x
region 450.
MITRAL
393
DENDRITES
dendrites as they extend horizontally from the mitral cell bodies. The section is parallel to and within the layer of mitral cell bodies, so that we are looking down on the layer from above, as it were. The dendritic trunks and branches form a tortuous meshwork running generally parallel to the plane of section; cross-cut dendrites are rare. These secondary dendrites are too tortuous and branched to be followed for any great distance but there appears to be an anteroposterior bias in the way the dendrites run, although it is difficult to characterize. More quantitative evidence for an anteroposterior bias in the orientation of the mitral secondary dendrites was obtained by comparing sections in different planes in the bulb. Figure 3 is from a frontal section, and shows
FIG.
bulbar
3. Frontal section, mid-anteroposterior in surface is above. Cresyl-violet. Magnification
bulb, dorsal X $50.
bulbar
region.
The
the external plexiform layer profusely pock-marked by small bodies. A few of these are clearly blood vessels; the rest are horizontally and tangentially running dendrites cut in cross section. In the deep part of the external plexiform layer, these are mostly mitral secondary dendrites. By comparison, in the longitudinal plane of Fig. I, the L’holes” in the section are few and many horizontal and tangential dendritic fragments can be seen running more or less in the plane of the section.
SHEPHERD
394
When the number of cross-cut dendrites per area of field was counted, the frontal sections through the deep half of the external plexiform layer consistently showed about three times the number seen in longitudinal sections. This difference was significant at the p < 0.01 level. The difference was seen when comparing the two planes of section in the same bulb and also when comparing the two planes of section in different bulbs (as in Figs. I and 3). While the density of cross-cut dendrites in frontal sections was more or less constant throughout the circumference of the bulb, the diameters tended to be smaller in the ventral than in the dorsal external plexiform layer. At the anterior pole of the bulb the three planes of sections showed no significant differences. Evidence has recently been presented that dendrodendritic synapses between mitral secondary dendrites and granule processes provide the pathway for the powerful recurrent inhibition of the mitral cells which has been demonstrated in microelectrode experiments (4). The mitral dendrites which mediate this inhibition have an anteroposterior orientation; they thus tend to parallel the mitral axons running deeper in the bulb, sending collaterals to the granule and external plexiform layers; they also parallel the numerous collaterals from the tufted cell axons running in an anteroposterior direction in the internal plexiform layer (5). One can thus conceive of the rabbit’s olfactory bulb as organized in longitudinal wedges, like the sections of an orange. The functional significance of this organization might be that it allows a spread of mitral inhibition in the anteroposterior direction, while restricting it in the mediolateral direction. Possibly this is a partial basis for Adrian’s (1) finding of an anteroposterior gradient in the intensities of the responses of mitral cells to certain odors. References 1. 2.
3.
4.
5.
D. 1953. Sensory messages and sensation. The response of the olfactory organ to different smells. Acta Physiol. Sand. 29: 5-14. CAMMERMEYER, J. 1961. The importance of avoiding “dark” neurons in experimental neuropathology. Acta Neuropathol. 1: 245-270. PHILLIPS, C. G., T. P. S. POWELL, and G. M. SHEPHERD. 1963. Responses of mitral cells to stimulation of the lateral olfactory tract in the rabbit, J. Physiol. London 166: 65-88. RALL, W., G. M. SHEPHERD, T. S. REESE, and M. W. BRIGHTMAN. 1966. Dendrodendritic synaptic pathway for inhibition in the olfactory bulb. Exptl. Neuuol. 14: 44-56. RAMON Y CAJAL, S. 1891. Origin y termination de las fibras nerviosas olfactorias. Gac. Sanit. Barcelona 3: 133-139; 174-181; 206-212.
ADRIAN,
E.
MITRAL
6.
7. 8.
DENDRITES
395
RAMON Y CAJAL, S. 1911. “Histologie du Systizme Nerveux de I’Homme et des Verthbrks,” 2. L. Azoulay [trans.] Maloine, Paris. SHEPHERD, G. M. 1963. Neuronal systems controlling mitral cell excitability. J. Physiol. London 166: 101-117. YAMAMOTO, C., T. YAMAMOTO, and K. IWAMA. 1963. The inhibitory systems in the olfactory bulb studied by intracellular recording. J. Neurophysiol. 26: 403-415.