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Biochemical evidence for uncrossed and crossed locus coeruleus projections to the spinal cord
Brain Research, 196 (1980) 237-241 © Elsevier/North-Holland Biomedical Press
237
Biochemical evidence for uncrossed and crossed locus coeruleus projections to the spinal cord
FAROUK KAROUM, J. W. COMMISSIONG, N. H. NEFF and R. J. WYATT Laboratory o] Clinical Psychopharmacology and (J. W.C. and N.H.N.) Laboratory of Preclinical Pharmacology, National Institute o f Mental Health, Saint Fdizabeths Hospital, Washington, D.C. 20032 (U.S.A.)
(Accepted April 17th, 1980) Key words: coerulospinal noradrenergic projection -- gas chromatography-mass spectrometry of
spinal and hippocampal catecholamines-- locus coeruleus lesion
Recent histochemicalS, 11 and biochemical 5 studies have revealed the existence of a major neuronal pathway projecting to the spinal cord from the locus coeruleus (LC) which contains norepinephrine (NE). It is unclear, however, whether this projection innervates the cord ipsilaterally and/or contralaterally. Therefore we studied the effects of unilateral electrolytic lesions of the LC on the distribution and metabolism of catecholamines in different regions of the cord. In this communication we present biochemical evidence for both crossed and uncrossed coerulospinal projections. Moreover, we provide confirmation for a discrete dopaminergic system in the spinal cord 4 and allude to the possible existence of such a system in the hippocampus z. Male Sprague-Dawley rats of 150-200 g, (Zivic-Miller Laboratories, Allison Park, Pa.) were anesthetized with chloral hydrate (400 mg/kg i.p.) and electrolytic lesions placed in the right LC using the procedures described previously 5. Rats were decapitated 10-14 days later and their spinal cords dissected out and placed on a glass plate cooled from below with dry ice. The spinal cords were sectioned into left and right halves by cutting through the posterior median sulcus and then into cervical, thoracic and lumbar regions. The hippocampi from the left and right sides of the brain were also removed for analysis. Dissection of the hippocampi was carefully performed to exclude other parts of the brain. Lesions were verified histologically as well as biochemically from the content of NE in the hippocampi from either side of the brain. The LC lesions included subcoeruleus cells. Norepinephrine, dopamine (DA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured by mass fragmentography as previously describedT-L Tissues were homogenized with 1 N HCI and the protein content 1° of the precipitated pellet used as the basis for comparision of samples. Unilateral lesion of the LC significantly reduced the NE content in the ipsilateral
5.9 ± 0.5 2.9 + 0.2** 5.3 A: 0.4 2.7 ± 0.4** 5.9 ± 0.7 2.8 ± 0.3**
Control cervical cord Cervical cord of lesioned rats
Control thoracic cord Thoracic cord o f lesioned rats
Control l u m b a r cord L u m b a r cord of lesioned rats 6.1 ± 0.8 3.5 ~: 0.4*
5.2 ± 0.4 4.6 ~ 0.9*
5.5 ± 0.3 3.8 ± 0.2*
3.7 ± 0.6 0.6 ± 0.1"
* P < 0.05 c o m p a r e d to the left side (paired t-test). ** P < 0.05 c o m p a r e d to the control on the s a m e side (independent t-test).
3.3 ± 0.9 2.8 ±_ 0.2
0.63 ± 0.06 0.50 ± 0.02
0.54 ± 0.09 0.54 :L_ 0.09
0.62 -+- 0.07 0.56 ± 0.09
0.26 _± 0.09 0.24 ± 0.02
Left
Left
Right
DA
ArE
Control h i p p o c a m p u s H i p p o c a m p u s of lesioned rats
Description
Values are in n g / m g protein ± S.E.M. 6 rats were included in each experiment.
0.48 ± 0.05 0.50 ± 0.2
0.56 ± 0.05 0.56 ± 0.05
0.56 ± 0.05 0.50 ± 0.04
0.22 ± 0.05 0.21 5_ 0.02
Right
NE, DA and MHPG content of the hippocampus and spinal cord after lesion of"the right locus coeruleus
TABLE I
2.0 1.1
~ 0.2 :i-0.1"*
1.5 2 0.1 0.77 ± 0.07**
1.6 ± 0.1 0.89 ± 0.12"*
Left
MHPG
2.0 % 0 . 1 1.5 ± 0.2*
1.5 -r 0.1 1.1 ± 0.1"
1.8 :_ 0.2 1.2 ± 0.2*
Right
t,O L.o
239 hippocampus as compared to the contralateral or the control hippocampus (Table I). Dopamine content remained unchanged. In contrast to the hippocampus, unilateral LC lesion significantly reduced NE and MHPC content in ipsilateral and contralateral sides of the cord in all regions examined (Table I). Apparently the metabolism of NE ipsilateral and contralateral to the LC lesion was not significantly altered by the lesion as the ratio of MHPG/NE for control and lesioned animals was about 0.3. These results indicate that there are crossed and uncrossed coerulospinal projections from the LC nuclei. Dopamine content of the cord remained unchanged after the lesion, providing added support for the presence of an independent dopaminergic system in the cord4. Based on the loss of NE content from the cord and assuming equal distribution of NE in all descending fibers and that the lesion did not alter NE metabolism, about 50% of the NE on each side of the cord originates from the contralateral LC. The ipsilateral LC apparently supplies about 30, 20 and 40% of NE, respectively, to the ipsilateral cervical, thoracic, and lumbar regions of the cord. Thus it appears that about 80, 70, and 90x, respectively, of the NE originates from the LC nuclei. Fig. 1 illustrates the NE distribution in the cord. These results are consistent with the histological studies of Nygren and Olson11 and of Commissiong et al.5 where it was observed that LC supplies a major portion of the NE all along the rat spinal cord. Furthermore, they are compatible with reports that catecholamine-containing fibers cross from side to side within the cords. In addition, they are consistent with the observation that contralateral stimulation of the LC facilitates the monosynaptic reflex in the lumbar cordle. .Hippocompus
, Locus Estimotion from
of
NE
oriQinoting
controloterol
LC
coeruleus
Estimation from
of
(LC) NE
ipsiloterol
Cervical
= 50%
Cervical
= 30%
Thorocic
= 50%
Thorocic
= 20%
Lumber
= 50%
Lumbar
originating LC
=. 40%
Fig. 1. Percentage NE assumed to originate from the contralateral and the ipsilateral LC. Values were estimated from data in Table I. For simplicity the diagram shows the descending fibers crossing contralaterally in the medullary region. Fiber crossing, however, might take place all along the spinal cords.
240 Norepinephrine-containing neurons of the hippocampus originate principally from the LC 1. Apparently most of the projections are uncrossed as a lesion of the right LC reduced N E content in the right hippocampus by about 80 ~; while there was no significant change in the left hippocampus, in contrast to NE, DA content did not change after an LC lesion, implying that there is a discrete dopaminergic innervation of the hippocampus. Dopamine in our hippocampal samples is probably not related 1o the presence of tissue from the subicular or entorhinal cortex as tissue punches from the dorsal hippocampus gave similar results (unpublished data). A discrete hippocampal DA system has been recently suggested by Bischoff et at. e. As illustrated in Fig. 1, about 301~ of the NE in the thoracic cord apparently does not originate in the LC nuclei. This observation is consistent with studies demonstrating that histochemical fluorescence of the sympathetic lateral columns is not lost following a lesion of the LC. The catecholamine innervation of the sympathetic lateral column most likely originates from A I - A 3 medullary nuclei described by Dahlstr6m and Fuxe a. While this manuscript was in preparation, Ad6r et al. 1 reported that neurons which originate in LC enter the spinal cord uncrossed, and that the LC does not supply a major portion of the catecholamine neurons to the cord. This report is in conflict with several studiesS, 11 as well as with our own findings. Placement of the lesions in the LC is of major importance and as has been previously shown, lesions in the vicinity but outside of the L C do not change spinal cord NE content when assayed histochemically or by gas chromatography-mass spectrometryS, H. Our samples were assayed by mass fragmentography after establishing absolute identity of the compounds by multiple ion monitoring. Ad~r et al. 1 used a fluorometric assay following column separation. The reason for the discrepancy remains to be elucidated. However, it is possible that some celt bodies of the LC and subcoeruleus project cranially and some caudally and very select lesions may discriminate between these cell body populations. This problem is now under study. In conclusion, unilateral lesions of the LC results in a major reduction of NE and M H P G content in ipsilateral and contralateral halves of the cervical, thoracic, and o/ lumbar regions of the spinal cord. NE content was decreased by about 0/o in the ipsilateral but not the contralateral hippocampus by unilateral LC lesions. Dopamine content of the spinal cord and hippocampus was unaltered by the lesions, suggesting separate dopaminergic neuronal systems in these tissues.
1 Ad6r, J. P., Postema, F. and Korf, J., Contribution of the locus coeruleus to the adrenergic innervation of the rat spinal cord: a biochemical study, J. neural Transm., 44 (1979) 159-173. 2 Bischoff, S., Scatton, B. and Korf, J., Biochemical evidence for a transmitter role of dopamine in the rat hippocampus, Brain Research, 165 (1979) 161-165. 3 Carlsson, A., Falck, B., Fuxe, K. and Hillarp, N.-A., Cellular localization of monoamines in the spinal cord, Acta physiol, scand., 60 (1964) 112-119. 4 Commissiong, J. W., Galli, C. L. and Nef, N. H., Differentiation of dopaminergic and noradrenergic neurons in rat spinal cord, J. Neurochem., 30 (1978) 1095-1099. 5 Commissiong, J, W., Hellstr6m, S. O. and Neff, N. H., A new projection from locus coeruleus to the spinal ventral columns: histochemical and biochemcial evidence, Brain Research, 148 (1978) 207-213.
241 6 DahlstrSm, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system 1. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55. 7 Karoum, F., Garrison, C. K., Neff, N. and Wyatt, R. J., Trans-synaptic modulation of dopamine metabolism in the rat superior cervical ganglion, J. Pharmacol. exp: Ther., 201 (1977) 654-66L 8 Karoum, F., Moyer-Schwing, J., Potkin, S. C. and Wyatt, R. J., Presence of free, sulfate and glucuronide conjugated 3-methoxy-4-hydroxyphenylglycol (MHPG) in human brain, cerebrospinal fluid and plasma, Brain Research, 125 (1977) 333-339. 9 Karoum, F., Speciale, Jr., S. G. and Wyatt, R. J., Biochemical characterization of rat sympathetic ganglion : Pharmacological effects of reserpine on ganglionic catecholamines, J. Pharmacol. exp. Ther., 211 (1979) 706-710. 10 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 11 Nygren, L.-G. and Olson, L. A., A new major projection from locus coeruleus: the main source of noradrenergic nerve terminals in the ventral and dorsal columns of the spinal cord, Brain Research, 132 (1977) 85-93. 12 Strahlendorf, J. C., Strahlendorf, H. K., Kingsley, R. E., Gintautas, J. and Barnes, C. D., Facilitation of the lumbar monosynaptic reflexes by locus coeruleus stimulation, Neuropharmacology, 19 (1980) 225-230.
237
Biochemical evidence for uncrossed and crossed locus coeruleus projections to the spinal cord
FAROUK KAROUM, J. W. COMMISSIONG, N. H. NEFF and R. J. WYATT Laboratory o] Clinical Psychopharmacology and (J. W.C. and N.H.N.) Laboratory of Preclinical Pharmacology, National Institute o f Mental Health, Saint Fdizabeths Hospital, Washington, D.C. 20032 (U.S.A.)
(Accepted April 17th, 1980) Key words: coerulospinal noradrenergic projection -- gas chromatography-mass spectrometry of
spinal and hippocampal catecholamines-- locus coeruleus lesion
Recent histochemicalS, 11 and biochemical 5 studies have revealed the existence of a major neuronal pathway projecting to the spinal cord from the locus coeruleus (LC) which contains norepinephrine (NE). It is unclear, however, whether this projection innervates the cord ipsilaterally and/or contralaterally. Therefore we studied the effects of unilateral electrolytic lesions of the LC on the distribution and metabolism of catecholamines in different regions of the cord. In this communication we present biochemical evidence for both crossed and uncrossed coerulospinal projections. Moreover, we provide confirmation for a discrete dopaminergic system in the spinal cord 4 and allude to the possible existence of such a system in the hippocampus z. Male Sprague-Dawley rats of 150-200 g, (Zivic-Miller Laboratories, Allison Park, Pa.) were anesthetized with chloral hydrate (400 mg/kg i.p.) and electrolytic lesions placed in the right LC using the procedures described previously 5. Rats were decapitated 10-14 days later and their spinal cords dissected out and placed on a glass plate cooled from below with dry ice. The spinal cords were sectioned into left and right halves by cutting through the posterior median sulcus and then into cervical, thoracic and lumbar regions. The hippocampi from the left and right sides of the brain were also removed for analysis. Dissection of the hippocampi was carefully performed to exclude other parts of the brain. Lesions were verified histologically as well as biochemically from the content of NE in the hippocampi from either side of the brain. The LC lesions included subcoeruleus cells. Norepinephrine, dopamine (DA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured by mass fragmentography as previously describedT-L Tissues were homogenized with 1 N HCI and the protein content 1° of the precipitated pellet used as the basis for comparision of samples. Unilateral lesion of the LC significantly reduced the NE content in the ipsilateral
5.9 ± 0.5 2.9 + 0.2** 5.3 A: 0.4 2.7 ± 0.4** 5.9 ± 0.7 2.8 ± 0.3**
Control cervical cord Cervical cord of lesioned rats
Control thoracic cord Thoracic cord o f lesioned rats
Control l u m b a r cord L u m b a r cord of lesioned rats 6.1 ± 0.8 3.5 ~: 0.4*
5.2 ± 0.4 4.6 ~ 0.9*
5.5 ± 0.3 3.8 ± 0.2*
3.7 ± 0.6 0.6 ± 0.1"
* P < 0.05 c o m p a r e d to the left side (paired t-test). ** P < 0.05 c o m p a r e d to the control on the s a m e side (independent t-test).
3.3 ± 0.9 2.8 ±_ 0.2
0.63 ± 0.06 0.50 ± 0.02
0.54 ± 0.09 0.54 :L_ 0.09
0.62 -+- 0.07 0.56 ± 0.09
0.26 _± 0.09 0.24 ± 0.02
Left
Left
Right
DA
ArE
Control h i p p o c a m p u s H i p p o c a m p u s of lesioned rats
Description
Values are in n g / m g protein ± S.E.M. 6 rats were included in each experiment.
0.48 ± 0.05 0.50 ± 0.2
0.56 ± 0.05 0.56 ± 0.05
0.56 ± 0.05 0.50 ± 0.04
0.22 ± 0.05 0.21 5_ 0.02
Right
NE, DA and MHPG content of the hippocampus and spinal cord after lesion of"the right locus coeruleus
TABLE I
2.0 1.1
~ 0.2 :i-0.1"*
1.5 2 0.1 0.77 ± 0.07**
1.6 ± 0.1 0.89 ± 0.12"*
Left
MHPG
2.0 % 0 . 1 1.5 ± 0.2*
1.5 -r 0.1 1.1 ± 0.1"
1.8 :_ 0.2 1.2 ± 0.2*
Right
t,O L.o
239 hippocampus as compared to the contralateral or the control hippocampus (Table I). Dopamine content remained unchanged. In contrast to the hippocampus, unilateral LC lesion significantly reduced NE and MHPC content in ipsilateral and contralateral sides of the cord in all regions examined (Table I). Apparently the metabolism of NE ipsilateral and contralateral to the LC lesion was not significantly altered by the lesion as the ratio of MHPG/NE for control and lesioned animals was about 0.3. These results indicate that there are crossed and uncrossed coerulospinal projections from the LC nuclei. Dopamine content of the cord remained unchanged after the lesion, providing added support for the presence of an independent dopaminergic system in the cord4. Based on the loss of NE content from the cord and assuming equal distribution of NE in all descending fibers and that the lesion did not alter NE metabolism, about 50% of the NE on each side of the cord originates from the contralateral LC. The ipsilateral LC apparently supplies about 30, 20 and 40% of NE, respectively, to the ipsilateral cervical, thoracic, and lumbar regions of the cord. Thus it appears that about 80, 70, and 90x, respectively, of the NE originates from the LC nuclei. Fig. 1 illustrates the NE distribution in the cord. These results are consistent with the histological studies of Nygren and Olson11 and of Commissiong et al.5 where it was observed that LC supplies a major portion of the NE all along the rat spinal cord. Furthermore, they are compatible with reports that catecholamine-containing fibers cross from side to side within the cords. In addition, they are consistent with the observation that contralateral stimulation of the LC facilitates the monosynaptic reflex in the lumbar cordle. .Hippocompus
, Locus Estimotion from
of
NE
oriQinoting
controloterol
LC
coeruleus
Estimation from
of
(LC) NE
ipsiloterol
Cervical
= 50%
Cervical
= 30%
Thorocic
= 50%
Thorocic
= 20%
Lumber
= 50%
Lumbar
originating LC
=. 40%
Fig. 1. Percentage NE assumed to originate from the contralateral and the ipsilateral LC. Values were estimated from data in Table I. For simplicity the diagram shows the descending fibers crossing contralaterally in the medullary region. Fiber crossing, however, might take place all along the spinal cords.
240 Norepinephrine-containing neurons of the hippocampus originate principally from the LC 1. Apparently most of the projections are uncrossed as a lesion of the right LC reduced N E content in the right hippocampus by about 80 ~; while there was no significant change in the left hippocampus, in contrast to NE, DA content did not change after an LC lesion, implying that there is a discrete dopaminergic innervation of the hippocampus. Dopamine in our hippocampal samples is probably not related 1o the presence of tissue from the subicular or entorhinal cortex as tissue punches from the dorsal hippocampus gave similar results (unpublished data). A discrete hippocampal DA system has been recently suggested by Bischoff et at. e. As illustrated in Fig. 1, about 301~ of the NE in the thoracic cord apparently does not originate in the LC nuclei. This observation is consistent with studies demonstrating that histochemical fluorescence of the sympathetic lateral columns is not lost following a lesion of the LC. The catecholamine innervation of the sympathetic lateral column most likely originates from A I - A 3 medullary nuclei described by Dahlstr6m and Fuxe a. While this manuscript was in preparation, Ad6r et al. 1 reported that neurons which originate in LC enter the spinal cord uncrossed, and that the LC does not supply a major portion of the catecholamine neurons to the cord. This report is in conflict with several studiesS, 11 as well as with our own findings. Placement of the lesions in the LC is of major importance and as has been previously shown, lesions in the vicinity but outside of the L C do not change spinal cord NE content when assayed histochemically or by gas chromatography-mass spectrometryS, H. Our samples were assayed by mass fragmentography after establishing absolute identity of the compounds by multiple ion monitoring. Ad~r et al. 1 used a fluorometric assay following column separation. The reason for the discrepancy remains to be elucidated. However, it is possible that some celt bodies of the LC and subcoeruleus project cranially and some caudally and very select lesions may discriminate between these cell body populations. This problem is now under study. In conclusion, unilateral lesions of the LC results in a major reduction of NE and M H P G content in ipsilateral and contralateral halves of the cervical, thoracic, and o/ lumbar regions of the spinal cord. NE content was decreased by about 0/o in the ipsilateral but not the contralateral hippocampus by unilateral LC lesions. Dopamine content of the spinal cord and hippocampus was unaltered by the lesions, suggesting separate dopaminergic neuronal systems in these tissues.
1 Ad6r, J. P., Postema, F. and Korf, J., Contribution of the locus coeruleus to the adrenergic innervation of the rat spinal cord: a biochemical study, J. neural Transm., 44 (1979) 159-173. 2 Bischoff, S., Scatton, B. and Korf, J., Biochemical evidence for a transmitter role of dopamine in the rat hippocampus, Brain Research, 165 (1979) 161-165. 3 Carlsson, A., Falck, B., Fuxe, K. and Hillarp, N.-A., Cellular localization of monoamines in the spinal cord, Acta physiol, scand., 60 (1964) 112-119. 4 Commissiong, J. W., Galli, C. L. and Nef, N. H., Differentiation of dopaminergic and noradrenergic neurons in rat spinal cord, J. Neurochem., 30 (1978) 1095-1099. 5 Commissiong, J, W., Hellstr6m, S. O. and Neff, N. H., A new projection from locus coeruleus to the spinal ventral columns: histochemical and biochemcial evidence, Brain Research, 148 (1978) 207-213.
241 6 DahlstrSm, A. and Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system 1. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta physiol, scand., 62, Suppl. 232 (1964) 1-55. 7 Karoum, F., Garrison, C. K., Neff, N. and Wyatt, R. J., Trans-synaptic modulation of dopamine metabolism in the rat superior cervical ganglion, J. Pharmacol. exp: Ther., 201 (1977) 654-66L 8 Karoum, F., Moyer-Schwing, J., Potkin, S. C. and Wyatt, R. J., Presence of free, sulfate and glucuronide conjugated 3-methoxy-4-hydroxyphenylglycol (MHPG) in human brain, cerebrospinal fluid and plasma, Brain Research, 125 (1977) 333-339. 9 Karoum, F., Speciale, Jr., S. G. and Wyatt, R. J., Biochemical characterization of rat sympathetic ganglion : Pharmacological effects of reserpine on ganglionic catecholamines, J. Pharmacol. exp. Ther., 211 (1979) 706-710. 10 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 11 Nygren, L.-G. and Olson, L. A., A new major projection from locus coeruleus: the main source of noradrenergic nerve terminals in the ventral and dorsal columns of the spinal cord, Brain Research, 132 (1977) 85-93. 12 Strahlendorf, J. C., Strahlendorf, H. K., Kingsley, R. E., Gintautas, J. and Barnes, C. D., Facilitation of the lumbar monosynaptic reflexes by locus coeruleus stimulation, Neuropharmacology, 19 (1980) 225-230.