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Differential distribution of locus coeruleus projections to the hippocampal formation: anatomical and biochemical evidence
366
Brain Resear~It, 325 ( lt*~.~) 3~t~ ~,~)
I l~cxtcJ BRE 20574
Differential distribution of locus coeruleus projections to the hippocampal formation: anatomical and biochemical evidence JOHN H HARING and JAMES N DAVIS Neurology Research Laboratory, VA Medtcal Center, Durham, NC 27705 and Departments of Medtcme (Neurology) and Pharmacology, Duke Umverstty, Durham, NC27710 (U S A.)
(Accepted August 28th, 1984) Key words" locus coemleus - - hlppocampal formation - - HRP - - degeneration - - HPLC
Locus coemleus (LC) fibers m the fornLxmainly innervate the septal pole of the dentate gyms, whereas the cmgulum projects to the ventral hippocampal formation to provide LC input to the dentate gyms. LC fibers tn the ventral amygdaloid bundle have a widespread distribution, innervating the entire hippocampal gyms, as well as the mid-septotemporal and temporal regions of the dentate gyms. The LC fibers of the fornix and ventral amygdaloid bundle do not appear to overlap significantlyin the septat pole of the dentate gyms. Lesions which remove locus coeruleus (LC) axons within the fornix and cingulum produce a partial noradrenergic denervation of the hippocampal formation. In response to this lesion, intact LC fibers within the ventral amygdaloid bundle (VAB) proliferate in an attempt to restore hippocampal noradrenergic innervation to normal2,3, 8. We have been studying the possibility that the observed axonal proliferation is mediated by collaterals of damaged axons. This response would be analogous to the reaction of a plant after pruning of its branches and has been identified by Schneider 12 as a characteristic of developing neurons. In order to support the 'pruning' hypothesis of LC compensatory axonal proliferation, single LC neurons would have to send collaterals to the hippocampat formation by either fornix or cingulum, as well as VAB. Moreover, only neurons whose collaterals have been damaged by the lesion would be expected to participate in the compensatory remnervation. Using retrograde double labeling techniques, we have obtained data which bolsters the hypothesis that pruning of collaterals induces the proliferation of LC axonsS,6. However, the present knowledge of the hippocampal distribution of LC afferents traveling in the
fornix, cmgulum, and VAB causes this interpretation of retrograde labeling to be more suggestive than definitive. The present study was designed to reinvestigate the distributions of LC fibers reaching the luppocampal formation by each of the 3 afferent pathways in order to more accurately interpret the results obtained from retrograde studies in light of our pruning hypothesis. Following electrolytic lesions of the septal region, two groups of rats received placements of horseradish peroxidase (HRP) in the dorsal dentate gyrus. One group (n = 10) had focal placements of H R P crystals H, limited to the septal pole of the dentate gyrus. A second group (n = 6) received a 0.2/zl injection of 30% H R P in saline which included the septal pole and mid-septotemporat region of the dentate gyrus. Transported H R P was visualized using tetramethyl benzidine as a chromogen 9. Electrolytic lesions of the cingulum were performed in 3 rats, and degenerating neural elements were stained by the method of Gallyas et al.4. Two weeks after lesions of either the septum or cingulum, analysis of hippocampal norepinephrine (NE) content was accomplished by high-pressure liquid chromatography of NE extracted from hippo-
Correspondence: J H Hanng, Department of Anatomy, St. Louis Umversxty,School of Medicine, 1402 South Grand Boulevard, St Louts, MO 63104, U S A 0006-8993/85/$03.30 (~ 1985 Elsevier Science Pubhshers B V
367 campal homogenates. In the case of rats with septal lesions (n = 8), each hippocampal formation was divided into dorsal and ventral halves and each half separated into dentate and hippocampal gyri. Hippocampi from animals with bilateral cingulum lesions (n = 6) were merely divided into septal, middle, and temporal thirds. Unlesioned animals (n = 6) served as controls, and superior cervical ganglionectomies were performed on all animals used in biochemical analyses1,8. In all animals with septal lesions and hippocampal HRP injections, retrograde labeling of entorhinal cortex and polymorphic neurons of the contralateral dentate gyrus was observed. In addition, anterograde labeling was apparent m mossy fibers, as well as in commissural and associational fibers arising from polymorphic cells of the dentate gyrus. However, cases having focal HRP placements limited to the septal third of the dentate gyrus yielded no HRP-positive neurons in either LC or the raphe nuclei. In contrast, large injections with enzyme spread into the mid-septotemporal hippocampal formation yielded labeling of neurons throughout the LC. These large injections did not result in the labeling of raphe neurons, however. In sections stained by the method of Gallyas et al. 4, degenerating fibers were seen in the cingulum distal to the site of damage. Caudal to the splenium, degenerating fibers looped around the corpus callosum to enter the retrospinal cortex. Cingulum fibers then traversed the hippocampal gyrus at the level of the hippocampal flexure and appeared to distribute diffusely in the temporal hippocampal formation. A difference in the terminal field densities of the cingulum projection to dentate and hippocampal gyri was not discernible in these preparations. In normal rats, NE levels were found to be about 3-fold greater in the dentate gyrus than in the hippocampal gyrus. Furthermore, the ventral dentate gyrus and ventral hippocampal gyrus contained 42% and 37% more NE, respectively, than their dorsal counterparts. Large lesions of the septal region produced a 30% decrease in the NE content of the hippocampal formation; however, these changes were mainly observed in the dentate gyrus. Levels of NE were significantly diminished (P < 0.01, Dunnette test) in both the dorsal and ventral dentate gyrus. The NE levels seen in the dorsal dentate gyrus were
reduced by approximately 25%, while a reduction of about 50% was obtained in the ventral dentate gyrus. In the hippocampal gyrus, dorsal NE levels were little affected by septal lesion, but a small, statistically insignificant, decrease was observed in the ventral hippocampus. Lesions of the cingulum alone yielded NE levels in the hippocampal formation which were about 27% lower than those of the control group. When the hippocampal formation was dissected into thirds along its septo-temporal axis, levels of NE were found to be very similar between the septal and middle thirds, although septal levels tended to be slightly higher than the mid-septotemporal region. In contrast, NE content in the temporal third was 2-fold greater than either the septal or middle thirds of the hippocampal formation. After lesions of the cingulum, little change was noted in the NE content of either the septal or middle thirds of the hippocampal formation. However, destruction of the cingulum sigmficantly reduced ventral hippocampal formation NE by approximately 50% compared to control values (P < 0.01, Dunnette test). The present study has produced several important observations concerning the innervation of the hippocampal formation by the LC. (1) Lesions on the cingulum resulted in a pattern of terminal degeneration largely limited to the ventral hippocampal formation. This anatomical observation was corroborated by the significant decline in NE content in the ventral third of the hippocampal formation after electrolytic destruction of the cingulum. (2) Large septal lesions, which presumably remove LC fibers destined for both cingulum and formx, produced a change in the normal pattern of LC labeling if the HRP injection was limited to the septal pole of the dentate gyrus. However, if the spread of enzyme extended into the mid-septotemporal hippocampal formation, the pattern of LC labeling seen after septal lesion resembled that observed after injections of the ventral dentate gyrus of normal animals. These data are thought to reflect the removal of formx input to the dorsal dentate gyrus, as well as the d~stnbution of VAB, which remained intact after the septal lesion. These data also indicate that little or no overlap exists between the LC fibers of fornix and VAB in the dorsal dentate gyrus. (3) After septal lesion, significant decreases in NE levels were seen only in the
368 dentate gyrus. This result ~s attributed to the removal of fornix and cingulum inputs to the dorsal and ventral dentate gyrus. The most striking finding of the present study is that the cingulum projects mainly to the ventral hippocampal formation where it supplies LC fibers to the dentate gyrus. Loy et al. 7 speculated that the cingulum could be providing LC input to this region; however, the prevalent opinion has been that the dorsal part of the hippocampal gyrus constitutes the target of LC axons traveling in the cmgulum2, 3,10. Since our anatomical and biochemical observations are clearly in agreement regarding cmgulum projections, ~t seems appropriate to redefine the target of LC axons which arrive in the hippocampal formation via the cingulum. In the context of the present redefinition of the cingulum distribution within the h~ppocampal formation, it is logical that the efficacy of our septal lesions be called to question. This is particularly relevant since the studies of Gage et al. 2.3 support
the concept of a dorsal hippocampal dmtnbut~on of LC fibers m the cmgulum. The observed 50% reduction in NE content of the ventral hlppocampal formation following lesion of either the septum or the cmgulum appears to support the concept that LC axons en route to the cingulum traverse the septum and are thus damaged in lesions of the type employed m the present study. The synthesis of these anatomical and biochemical observations suggests a differential distribution of the three LC pathways to the hippocampal formation (Fig. 1). In our proposed scheme, LC fibers m the formx primarily innervate the septal pole of dentate gyrus, whereas the main target of the cingulum is the ventral dentate gyrus. VAB seems to have the most widespread distribution of the three pathways. LC fibers in VAB appear to innervate the entire hippocampal gyrus, as well as the mid-septotemporal and ventral regions of the dentate gyms. As previously noted, little overlap occurs between LC fibers of the
I::OI:N× ~tJId
/ t
/
\
/
\
,/ I/
\',\
Fig. 1 Diagrammatic summary of the distribution of locus coeruleus inputs to the htppocampal formation Drawing modified from Loy et al.7
369 fornix and those of V A B . Conversely, there is significant overlap b e t w e e n V A B and cingulum inputs in the ventral dentate gyrus. The distribution of LC afferent pathways suggested by the present study supports previous observations of differential LC label-
1 Crutcher, K A., Brothers, L and Davis, J. N , Sympathetic noradrenerglc sprouting m response to central cholinergic denervation, a hlstochemicai study of neuronal sprouting in the rat hippocampal formation, Bram Research, 210 (1981) 115-128 2 Gage, F H., Blorklund, A. and Stenevi, U., Relnnervation of the parually deafferented hippocampus by compensatory collateral sprouting from spared cholinerglc and noradrenergic afferents, Brain Research, 268 (1983) 27-37. 3 Gage, F H , Bjorklund, A. and Stenevl, U., Local regulation of compensatory noradrenergic hyperactivity m the partially denervated bippocampus, Nature (Lond.), 303 (1983) 819-821 4 Gallyas, F., Wolff, J R., Bottcher, H. and Zaborsky, L., A reliable and sensitwe method to locate terminal degeneration and lysosomes in the CNS, Stare Technol., 55 (1980) 299-306. 5 Haring, J H. and Davis, J. N., Topography of locus coeruleus neurons projecting to the area dentata, Exp. Neurol., 79 (1983) 785-800. 6 Haring, J. H and Davis, J N., Only collaterals of injured locus coeruleus fibers participate in compensatory axonal prohferatlon, Anat Rec., 208 (1984) 70A. 7 Loy, R. D , Koziell, A , Lindsey, J. A and Moore, R. Y.,
ing5 and lends credence to our contention that pruning induces compensatory proliferation of LC axons 6. Supported by N I H G r a n t NS 06233.
Noradrenergic innervatlon of the adult rat hlppocampal formation, J. comp Neurol., 189 (1980) 699-710. 8 Madison, R. and Davis, J. N., Sprouting of noradrenergic fibers m hlppocampus following medial septal lesions: contnbutions of the central and peripheral nervous systems, Exp. Neurol., 80 (1983) 167-177 9 Mesulam, M.-M., Tetramethyl benzidlne for horseradish peroxldase neurohistochemlstry, a non-carcmogemc blue reaction with superior sensitivity for vasuahzing neural afferents and efferents, J. Htstochem. Cytochem, 26 (1978) 106-117 10 Moore, R. Y. and Bloom, F. E., Central catecholamine neuron systems: anatomy and physiology of the norepinephnne and epinephnne systems, Ann Rev. Neurosct., 2 (1979) 113-168. 11 Mort, J., Horl, H. and Katsuda, N., A new method for application of horseradish peroxldase into a restricted area of the brain, Brain Res Bull., 6 (1981) 19-22 12 Schneider, G. E. and Jhavan, S R., Neuroanatomlcal correlates of spared or altered function after brain lesions in the newborn hamster In D. G. Stem, J. J Rosen and N. Butters (Eds), Plasticity and Recovery of Functton in the Central Nervous System, Academic Press, New York, 1974, pp. 65-110.
Brain Resear~It, 325 ( lt*~.~) 3~t~ ~,~)
I l~cxtcJ BRE 20574
Differential distribution of locus coeruleus projections to the hippocampal formation: anatomical and biochemical evidence JOHN H HARING and JAMES N DAVIS Neurology Research Laboratory, VA Medtcal Center, Durham, NC 27705 and Departments of Medtcme (Neurology) and Pharmacology, Duke Umverstty, Durham, NC27710 (U S A.)
(Accepted August 28th, 1984) Key words" locus coemleus - - hlppocampal formation - - HRP - - degeneration - - HPLC
Locus coemleus (LC) fibers m the fornLxmainly innervate the septal pole of the dentate gyms, whereas the cmgulum projects to the ventral hippocampal formation to provide LC input to the dentate gyms. LC fibers tn the ventral amygdaloid bundle have a widespread distribution, innervating the entire hippocampal gyms, as well as the mid-septotemporal and temporal regions of the dentate gyms. The LC fibers of the fornix and ventral amygdaloid bundle do not appear to overlap significantlyin the septat pole of the dentate gyms. Lesions which remove locus coeruleus (LC) axons within the fornix and cingulum produce a partial noradrenergic denervation of the hippocampal formation. In response to this lesion, intact LC fibers within the ventral amygdaloid bundle (VAB) proliferate in an attempt to restore hippocampal noradrenergic innervation to normal2,3, 8. We have been studying the possibility that the observed axonal proliferation is mediated by collaterals of damaged axons. This response would be analogous to the reaction of a plant after pruning of its branches and has been identified by Schneider 12 as a characteristic of developing neurons. In order to support the 'pruning' hypothesis of LC compensatory axonal proliferation, single LC neurons would have to send collaterals to the hippocampat formation by either fornix or cingulum, as well as VAB. Moreover, only neurons whose collaterals have been damaged by the lesion would be expected to participate in the compensatory remnervation. Using retrograde double labeling techniques, we have obtained data which bolsters the hypothesis that pruning of collaterals induces the proliferation of LC axonsS,6. However, the present knowledge of the hippocampal distribution of LC afferents traveling in the
fornix, cmgulum, and VAB causes this interpretation of retrograde labeling to be more suggestive than definitive. The present study was designed to reinvestigate the distributions of LC fibers reaching the luppocampal formation by each of the 3 afferent pathways in order to more accurately interpret the results obtained from retrograde studies in light of our pruning hypothesis. Following electrolytic lesions of the septal region, two groups of rats received placements of horseradish peroxidase (HRP) in the dorsal dentate gyrus. One group (n = 10) had focal placements of H R P crystals H, limited to the septal pole of the dentate gyrus. A second group (n = 6) received a 0.2/zl injection of 30% H R P in saline which included the septal pole and mid-septotemporat region of the dentate gyrus. Transported H R P was visualized using tetramethyl benzidine as a chromogen 9. Electrolytic lesions of the cingulum were performed in 3 rats, and degenerating neural elements were stained by the method of Gallyas et al.4. Two weeks after lesions of either the septum or cingulum, analysis of hippocampal norepinephrine (NE) content was accomplished by high-pressure liquid chromatography of NE extracted from hippo-
Correspondence: J H Hanng, Department of Anatomy, St. Louis Umversxty,School of Medicine, 1402 South Grand Boulevard, St Louts, MO 63104, U S A 0006-8993/85/$03.30 (~ 1985 Elsevier Science Pubhshers B V
367 campal homogenates. In the case of rats with septal lesions (n = 8), each hippocampal formation was divided into dorsal and ventral halves and each half separated into dentate and hippocampal gyri. Hippocampi from animals with bilateral cingulum lesions (n = 6) were merely divided into septal, middle, and temporal thirds. Unlesioned animals (n = 6) served as controls, and superior cervical ganglionectomies were performed on all animals used in biochemical analyses1,8. In all animals with septal lesions and hippocampal HRP injections, retrograde labeling of entorhinal cortex and polymorphic neurons of the contralateral dentate gyrus was observed. In addition, anterograde labeling was apparent m mossy fibers, as well as in commissural and associational fibers arising from polymorphic cells of the dentate gyrus. However, cases having focal HRP placements limited to the septal third of the dentate gyrus yielded no HRP-positive neurons in either LC or the raphe nuclei. In contrast, large injections with enzyme spread into the mid-septotemporal hippocampal formation yielded labeling of neurons throughout the LC. These large injections did not result in the labeling of raphe neurons, however. In sections stained by the method of Gallyas et al. 4, degenerating fibers were seen in the cingulum distal to the site of damage. Caudal to the splenium, degenerating fibers looped around the corpus callosum to enter the retrospinal cortex. Cingulum fibers then traversed the hippocampal gyrus at the level of the hippocampal flexure and appeared to distribute diffusely in the temporal hippocampal formation. A difference in the terminal field densities of the cingulum projection to dentate and hippocampal gyri was not discernible in these preparations. In normal rats, NE levels were found to be about 3-fold greater in the dentate gyrus than in the hippocampal gyrus. Furthermore, the ventral dentate gyrus and ventral hippocampal gyrus contained 42% and 37% more NE, respectively, than their dorsal counterparts. Large lesions of the septal region produced a 30% decrease in the NE content of the hippocampal formation; however, these changes were mainly observed in the dentate gyrus. Levels of NE were significantly diminished (P < 0.01, Dunnette test) in both the dorsal and ventral dentate gyrus. The NE levels seen in the dorsal dentate gyrus were
reduced by approximately 25%, while a reduction of about 50% was obtained in the ventral dentate gyrus. In the hippocampal gyrus, dorsal NE levels were little affected by septal lesion, but a small, statistically insignificant, decrease was observed in the ventral hippocampus. Lesions of the cingulum alone yielded NE levels in the hippocampal formation which were about 27% lower than those of the control group. When the hippocampal formation was dissected into thirds along its septo-temporal axis, levels of NE were found to be very similar between the septal and middle thirds, although septal levels tended to be slightly higher than the mid-septotemporal region. In contrast, NE content in the temporal third was 2-fold greater than either the septal or middle thirds of the hippocampal formation. After lesions of the cingulum, little change was noted in the NE content of either the septal or middle thirds of the hippocampal formation. However, destruction of the cingulum sigmficantly reduced ventral hippocampal formation NE by approximately 50% compared to control values (P < 0.01, Dunnette test). The present study has produced several important observations concerning the innervation of the hippocampal formation by the LC. (1) Lesions on the cingulum resulted in a pattern of terminal degeneration largely limited to the ventral hippocampal formation. This anatomical observation was corroborated by the significant decline in NE content in the ventral third of the hippocampal formation after electrolytic destruction of the cingulum. (2) Large septal lesions, which presumably remove LC fibers destined for both cingulum and formx, produced a change in the normal pattern of LC labeling if the HRP injection was limited to the septal pole of the dentate gyrus. However, if the spread of enzyme extended into the mid-septotemporal hippocampal formation, the pattern of LC labeling seen after septal lesion resembled that observed after injections of the ventral dentate gyrus of normal animals. These data are thought to reflect the removal of formx input to the dorsal dentate gyrus, as well as the d~stnbution of VAB, which remained intact after the septal lesion. These data also indicate that little or no overlap exists between the LC fibers of fornix and VAB in the dorsal dentate gyrus. (3) After septal lesion, significant decreases in NE levels were seen only in the
368 dentate gyrus. This result ~s attributed to the removal of fornix and cingulum inputs to the dorsal and ventral dentate gyrus. The most striking finding of the present study is that the cingulum projects mainly to the ventral hippocampal formation where it supplies LC fibers to the dentate gyrus. Loy et al. 7 speculated that the cingulum could be providing LC input to this region; however, the prevalent opinion has been that the dorsal part of the hippocampal gyrus constitutes the target of LC axons traveling in the cmgulum2, 3,10. Since our anatomical and biochemical observations are clearly in agreement regarding cmgulum projections, ~t seems appropriate to redefine the target of LC axons which arrive in the hippocampal formation via the cingulum. In the context of the present redefinition of the cingulum distribution within the h~ppocampal formation, it is logical that the efficacy of our septal lesions be called to question. This is particularly relevant since the studies of Gage et al. 2.3 support
the concept of a dorsal hippocampal dmtnbut~on of LC fibers m the cmgulum. The observed 50% reduction in NE content of the ventral hlppocampal formation following lesion of either the septum or the cmgulum appears to support the concept that LC axons en route to the cingulum traverse the septum and are thus damaged in lesions of the type employed m the present study. The synthesis of these anatomical and biochemical observations suggests a differential distribution of the three LC pathways to the hippocampal formation (Fig. 1). In our proposed scheme, LC fibers m the formx primarily innervate the septal pole of dentate gyrus, whereas the main target of the cingulum is the ventral dentate gyrus. VAB seems to have the most widespread distribution of the three pathways. LC fibers in VAB appear to innervate the entire hippocampal gyrus, as well as the mid-septotemporal and ventral regions of the dentate gyms. As previously noted, little overlap occurs between LC fibers of the
I::OI:N× ~tJId
/ t
/
\
/
\
,/ I/
\',\
Fig. 1 Diagrammatic summary of the distribution of locus coeruleus inputs to the htppocampal formation Drawing modified from Loy et al.7
369 fornix and those of V A B . Conversely, there is significant overlap b e t w e e n V A B and cingulum inputs in the ventral dentate gyrus. The distribution of LC afferent pathways suggested by the present study supports previous observations of differential LC label-
1 Crutcher, K A., Brothers, L and Davis, J. N , Sympathetic noradrenerglc sprouting m response to central cholinergic denervation, a hlstochemicai study of neuronal sprouting in the rat hippocampal formation, Bram Research, 210 (1981) 115-128 2 Gage, F H., Blorklund, A. and Stenevi, U., Relnnervation of the parually deafferented hippocampus by compensatory collateral sprouting from spared cholinerglc and noradrenergic afferents, Brain Research, 268 (1983) 27-37. 3 Gage, F H , Bjorklund, A. and Stenevl, U., Local regulation of compensatory noradrenergic hyperactivity m the partially denervated bippocampus, Nature (Lond.), 303 (1983) 819-821 4 Gallyas, F., Wolff, J R., Bottcher, H. and Zaborsky, L., A reliable and sensitwe method to locate terminal degeneration and lysosomes in the CNS, Stare Technol., 55 (1980) 299-306. 5 Haring, J H. and Davis, J. N., Topography of locus coeruleus neurons projecting to the area dentata, Exp. Neurol., 79 (1983) 785-800. 6 Haring, J. H and Davis, J N., Only collaterals of injured locus coeruleus fibers participate in compensatory axonal prohferatlon, Anat Rec., 208 (1984) 70A. 7 Loy, R. D , Koziell, A , Lindsey, J. A and Moore, R. Y.,
ing5 and lends credence to our contention that pruning induces compensatory proliferation of LC axons 6. Supported by N I H G r a n t NS 06233.
Noradrenergic innervatlon of the adult rat hlppocampal formation, J. comp Neurol., 189 (1980) 699-710. 8 Madison, R. and Davis, J. N., Sprouting of noradrenergic fibers m hlppocampus following medial septal lesions: contnbutions of the central and peripheral nervous systems, Exp. Neurol., 80 (1983) 167-177 9 Mesulam, M.-M., Tetramethyl benzidlne for horseradish peroxldase neurohistochemlstry, a non-carcmogemc blue reaction with superior sensitivity for vasuahzing neural afferents and efferents, J. Htstochem. Cytochem, 26 (1978) 106-117 10 Moore, R. Y. and Bloom, F. E., Central catecholamine neuron systems: anatomy and physiology of the norepinephnne and epinephnne systems, Ann Rev. Neurosct., 2 (1979) 113-168. 11 Mort, J., Horl, H. and Katsuda, N., A new method for application of horseradish peroxldase into a restricted area of the brain, Brain Res Bull., 6 (1981) 19-22 12 Schneider, G. E. and Jhavan, S R., Neuroanatomlcal correlates of spared or altered function after brain lesions in the newborn hamster In D. G. Stem, J. J Rosen and N. Butters (Eds), Plasticity and Recovery of Functton in the Central Nervous System, Academic Press, New York, 1974, pp. 65-110.