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Growth of medial forebrain bundle axons into peripheral nerve grafts in the rat
198
Brain Research, 372 (1986) 198-203 Elsevier
BRE 21531
Growth of medial forebrain bundle axons into peripheral nerve grafts in the rat RICHARD P. DUM and CAMILLE G. SALAME Department of Neurosurgery, State University of New York at Upstate Medical Center, Syracuse, NY 13210 (U.S.A.)
(Accepted January 14th, 1986) Key words: regeneration - - medial forebrain bundle - - monoaminergic neuron - - nerve graft
In rats, we intercepted medial forebrain bundle axons just lateral to the hypothalamus with peripheral nerve grafts which terminated extracranially. The neurons which grew into the nerve grafts were labeled with retrogradely transported fluorescent dyes. Catecholamines were labeled with glyoxylic acid histofluorescence. Most nuclei, particularly the raphe complex and locus coeruleus, which project rostraily into the medial forebrain bundle were labeled. Many catecholamine fibers were observed in the graft even after removal of the superior cervical ganglions. Thus, monoaminergic neurons which were located relatively remotely from the implant site exhibited rather selective regrowth into the nerve grafts.
Many different types of central nervous system (CNS) neurons have demonstrated the ability to sprout and elongate into implanted peripheral nerve grafts in rats 1'2's'23'24. The axonal growth into these grafted nerves is usually limited, however, to neurons rather close to the site of the implant. This characteristic of the regrowing neuronal population imposes at least two limitations on the physiological evaluation of these regrowing neurons. First, the diversity of regrowing neurons decreases the probability of a homogeneous effect on potential target neurons. Second, the graft must be implanted so close to the desired neuronal population that their normal morphology and, hence, function may be significantly altered. We have minimized these problems by implanting a nerve graft to intercept monoaminergic axons coursing in the medial forebrain bundle (MFB) 4'5'10'20. This graft location induced a more homogeneous population of neurons to grow without disrupting their normal nuclear morphology. Under aseptic conditions, a small burr hole was made in the skull (1.5 m m lateral, +3.5 mm interaural) of young adult female rats (200 g) which were anesthetized with chloral hydrate (400 mg/kg). A 30-mm piece of autologous sciatic nerve was removed, wrapped around the end of a glass capillary
tube and plunged vertically to the base of the brain. The graft was targeted to interrupt the medial forebrain bundle at the caudal border of the hypothalamus 4. The free end of the graft was led externally for about 15 mm and sutured into the temporalis muscle. Up to one year later, each rat was reanesthetized and the distal end of the graft was mobilized and transected. The cut end was placed on a rubber sheet, surrounded with vaseline and soaked in a tracer substance dissolved in 2% dimethylsulfoxide (DMSO) 8. The tracer substance was one of the following fluorescent dyes: Evans blue (EB), Diamidino yellow (DY), or a mixture of Granular blue (GB) plus horseradish peroxidase (HRP) 3'14'16. The DY treated rat had its superior cervical ganglions removed bilaterally at this time 25. Five days later, GB + H R P treated rats were perfused intracardially with saline, 10% buffered formalin and 10% buffered formalin with 10% glycerin. These brains were blocked and sectioned frozen at 30 ktm. Alternate sections were reacted for H R P by the tetramethyl benzidine method 17. EB- and DY-treated rats were pretreated with Nialamide (400 mg/kg, i.p.) 6 h prior to sacrifice 7. The rats were anesthetized and their brains were removed whole and frozen. Cryostat sections were cut at 8 / t m and reacted by the glyoxylic acid
Correspondence: R.P. Dum, 3118 Weiskotten Hall, 766 Irving Avenue, Syracuse, NY 13210, U.S.A.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
199
A
B250 (.h ._/ .._I t,l
150 I.I.
o ,"r t,l m
:E :m. 50
F t 0
1.8
A
A 3.6
DISTANCE
(ram)
5,4
C
( ! B
4
Imm
Fig. 1. Location and distribution of GB-labeled neurons whose axons grew into a peripheral nerve bridge inserted through the cortex, thalamus and hypothalamus. A: trajectory of graft through thalamus and hypothalamus. Cortex omitted from diagram. B: histogram illustrating the rostrocaudal distribution of labeled neurons. Shaded column indicates location of graft. Arrows correspond to locations of the 3 sections appearing in C in the appropriate left-to-right sequence. C: drawings of 3 sections showing the location of labeled neurons.
method for catecholamine (CA) histofluorescence 9. Every fourth (GB) or sixth (EB, DY) section was examined for fluorescently labeled neurons using a Zeiss microscope with mercury lamp and the 365-nm (GB), 405-nm (CA, DY) and 510-560-nm (EB) filter packs. The labeled neurons were plotted on an X-Y plotter connected to the microscope stage. Nuclear locations and landmarks were established by subsequent counterstaining of the same sections with cresyl violet. Five of the 7 surviving experimental rats exhibited excellent retrograde transport and neuronal labeling. In these cases, the nerve graft intercepted the MFB either at the border between the ventral tegmental area and the substantia nigra or more rostrally in the lateral hypothalamus in the region just medial to the cerebral peduncle and subthalamus (Fig. 1A) 12'18'22. From its termination in the hypothalamic area, the
graft extended dorsally through the thalamus and exited from the cortex. Rostral to the graft, we observed only sparse CA terminal labeling within the ipsilateral hypothalamus. Just caudal to the graft, we found dense accumulations of CA in the area of the MFB on the ipsilateral side as compared to the unoperated side (Fig. 2D). Such dense accumulations of CA have been observed to characterize the delicate, closely packed sprouts of CA fibers which remained for at least 6 months following transection of the MFB 4. These observations provided independent confirmation that the nerve graft did indeed intercept and transect many of the CA axons in the MFB. The location of labeled neurons exhibited a consistent pattern (Fig. 1C, Table I). Nearly all the labeled neurons were caudal to the graft and were located in nuclei which project rostrally into the MFB 26. The
200 raphe complex and locus coeruleus (LC) were always labeled (Table I). The dorsal raphe (DR; Figs. 1C,
2B) contained at least 50% of all labeled cells while the median raphe, raphe pontis and caudal linear nu-
Fig. 2. Photomicrographs of GB-labeled neurons in the dorsal raphe (A, B) and catecholamine fibers in the graft (C) and the MFB (D). A: photomicrograph of GB-labeled neurons in neck of dorsal raphe. Calibration bar, 50/~m. B: photomicrograph of GB-labeled cells primarily in the ipsilateral dorsal raphe. Aqueduct visible at top. Medial longitudinal fasciculivisible on either side of the neck of the DR at bottom. Calibration bar, 200/~m. C: catecholamine-labeled fibers within graft. Bright fibers near top of the photomicrograph delineate the edge of graft. Calibration bar, 25/~m. D: photomicrograph of basal hypothalamus showing dense catecholamine histofluorescence around the MFB just caudal to the graft (left) vs a very sparse amount on the control side (right). The large, dark oval outline of a blood vessel marks the midline.
201 TABLE I Number and location of labeled CNS neurons following application of retrograde tracer substances to the distal end of the peripheral nerve graft implanted in the diencephalon of the rat Nucleus
BBR2 BBR5 BBR6 BBR7 BBRIO
Noradrenergic nuclei A2 0 locus coeruleus 112 parabrachial 4 Raphe nuclei dorsal raphe 549 raphe pontis 35 median raphe 241 caudal linear 36 Tegmental nuclei lateral and dorsal 0 ventral 6 pontine reticular 2 ventral tegmental area 0 interpeduncular 16 pontine central gray 0 hypothalamus 67 pontine and medullary RF 16 zona incerta 0 entopeduncular 0 cortex 0 motor nucleus of 71 0 Total
1084
6 186 0
0 234 0
0 9 0
0 102 11
408 0 30 60
474 36 118 18
146 11 63 17
535 27 63 20
0 0 0
8 0 0
0 1 0
8 0 0
0 0 0 0
6 10 8 0
6 12 0 6
54 0 0 11
0 0 0 0 0
44 0 0 0 132
1 0 0 0 0
3 118 43 1 0
272
995
690
9562
1 Motor nucleus of 7 was probably labeled by fluorescent dye spilled on the terminations of the auriculopalpebralis branch of the 7th nerve and not by growth of motoneurons into the graft within the brain. 2 Total does not include cells in the 7th nucleus.
clei were consistently labeled with significant numbers of neurons. The labeling tended to be ipsilateral although this distinction was somewhat obscured in the midline raphe nuclei (Figs. 1C, 2B). Outside of the MFB nuclei, the hypothalamic, zona incerta and entopeduncular nuclei near the central end of the graft infrequently contained labeled neurons. In particular, only one labeled neuron was ever found adjacent to the graft as it coursed through the cortex and thalamus. Large numbers of neurons were labeled (mean 800 + 330; range 272-1084) in each rat. In contrast to several prior studies 1'8'23'24, most of the regrowing neurons were at least 1.8 mm distant from the cut end of the graft. The typical distribution was bimodal (Fig. 1B), reflecting the large number of neurons in the D R and LC, respectively.
Because most of the labeled neurons arose from nuclei which contain significant monoaminergic populations 7, 3 rat brains were reacted with glyoxylic acid to produce C A histofluorescence in order to directly determine whether CA-containing neurons were growing into the graft. Nearly all of the neurons within LC and A2 which were labeled with EB or D Y were also clearly fluorescent for CA. Even after the removal of both superior cervical ganglions, a diffuse bright cloud of C A fluorescence was observed within the initial segment of the central end of the graft. As the graft continued dorsally through the thalamus, individual C A fibers emerged from the cloud of C A fluorescence, ran parallel to its long axis, and continued within the graft as it exited the cranium (Fig. 2C). However, no fibers were seen within or crossing the epineurium. These observations strongly support the growth of C A axons into the cut central end of the graft. To ensure that the pattern of labeling was not simply due to spillage or leakage of dye onto the cortex, we placed dye directly on rat cortex as a control. Direct application of dye to the cortex produced heavy labeling of cortical neurons at the application site. In contrast, only one labeled cortical neuron was observed in 7 experimental rats, two of which did not exhibit any neuronal labeling. Therefore, any direct spillage of dye onto the cortex would have been easily detected. Our results demonstrated the ability of many neurons which projected within the MFB to grow into peripheral nerve grafts in the rat. Two features of this regrowing neuronal population were of particular interest. First, there was rather selective regrowth of primarily monoaminergic neurons into the nerve graft. Second, most of the regrowing neurons were located relatively remotely from the site of the graft implant. The growth of a specific neuronal population was surprising in view of the fact that most previous reports 1'8'23'24found non-specific ingrowth primarily from neurons adjacent to the graft and a quite precipitous decline in numbers at greater distances. Selective regrowth into nerve grafts may have depended on several factors. First, the type of neuron, as defined by its neurotransmitter, may have been a determining factor 2. The vast majority of our labeled neurons came from nuclei which contain monoaminergic neurotransmitters 7'2~. In particular, nearly all
202 of the neurons in LC which were labeled from the graft were found to contain cateeholamines. Because many of the neurons in the heavily labeled D R and deeper midline raphe nuclei have serotonin as a neurotransmitter TM, some serotonergic neurons were probably among those which projected into the graft. Monoaminergic neurons have demonstrated remarkable abilities to sprout collaterals 1°, to regenerate after chemical lesions 6, to sprout after transection 5'13 and to grow into iris and mitral valve implanted in the brain 4. A number of reasons have been proposed for this remarkable regenerative capacity including unmyelinated fibers 4, their old phylogeny 15 and their ubiquitous, non-specific termination pattern which suggested a neuromodulatory role rather than one for the transmission of specific information 19. A second reason for the rather selective regrowth of MFB neurons into the graft may have been that entry of the regrowing axons was restricted to the cut (open) end of the graft. Such restrictions appeared to apply in regenerating peripheral nerves. Regenerating peripheral axons sprouted into and elongated within the basal lamina tubes containing proliferating Schwann cells 27. Assuming that the same conditions were required for central nervous system axons to elongate within a peripheral nerve graft, the cortical and thalamic neurons located along the course of the nerve graft would have been denied access to open Schwann cell tubes. A related factor was that the epineurium-perineurium sheathing of the nerve graft or glial scar formation in response to the implantation may have provided a sufficient barrier to prevent the entry of CNS axons along the course of the graft. The absence of labeled neurons located along the course of the graft within the cortex and thalamus was not
1 Benfey, M. and Aguayo, A.J., Extensive elongation of axons from rat brain into peripheral nerve grafts, Nature (London), 296 (1982) 150-152. 2 Benfey, M., Bringer, U.R., Vidal-Sanz, M., Bray, G.M. and Aguayo, A.J., Axonal regeneration from GABAergic neurons in the adult rat thalamus, J. Neurocytol., 14 (1985) 279-296. 3 Bentivoglio, M., Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Dann, O., Fluorescent retrograde neuronal labeling in rats by means of substances binding specifically to adenine-thymine rich DNA, Neurosci. Lett., 12 (1979) 235-240. 4 Bj6rklund, A. and Stenevi, U., Growth of central catechol-
due to an intrinsic inability of these neurons to regrow. Some neurons within this region have demonstrated the capacity to grow into peripheral nerve grafts 12. A third factor may have been that the central end of the graft always intercepted and presumably interrupted the axons of neurons contributing to the MFB. Although the labeled neurons almost always projected within the MFB, several nuclei which contribute to the MFB were not labeled in our experiments. MFB nuclei which only project into the caudal part of the MFB 26 were unlabeled or sparsely labeled. These nuclei included the following: A1, A2, parabrachial, dorsal and lateral tegmental, and ventral tegmental area. Their axons could have escaped transection. The raphe magnus nucleus projects through the MFB but is located far from the implant site. The absence of labeled cells in this nucleus may reflect the sharply decreasing probability of regrowth which occurs as the distance between the graft and cell body location increases 1's'23'24. Thus, both the presence and the degree of axonal damage may be prerequisites for the initiation of regenerative growth 8,23. The selective regrowth of primarily monoaminergic neurons provides a new opportunity to determine whether these newly growing axons can form functional synapses. It should be possible to implant a graft between the MFB and a denervated hippocampus in order to determine whether monoaminergic reinnervation and behavioral improvement occurs It. Because most of the regrowing neurons are located relatively remotely from the site of the graft implant, only minimal disruption of the normal nuclear morphology and hence functional output should occur.
amine neurons into smooth muscle grafts in the rat mesencephalon, Brain Research, 31 (1971) 1-20. 5 Bj6rklund, A., Katzman, R., Stenevi, U. and West, K.A., Development and growth of axonal sprouts from noradrenaline and 5-hydroxytryptamine neurons in the rat spinal cord, Brain Research, 31 (1971) 21-33. 6 BjOrklund, A. and Lindvall, O., Regeneration of normal terminal innervation patterns by central noradrenergic neurons after 5,7-dihydroxytryptamine induced axotomy in the adult rat, Brain Research, 171 (1979) 271-293. 7 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine containing neurons in the central nervous system, Acta Physiol. Scand., 62, Suppl. 232 (1964) 1-55.
203 8 David, S. and Aguayo, A.J., Axonal elongation into peripheral nervous system 'bridges' after central nervous system injury in adult rats, Science, 214 (1981) 931-933. 9 De la Torre, J.C. and Surgeon, J.W., A methodological approach to rapid and sensitive monoamine histofluorescence using a modified glyoxylic acid technique: the SPG method, Histochemistry, 49 (1976) 81-93. 10 Gage, F.H., Bj6rklund, A. and Stenevi, U., Reinnervation of the partially deafferented hippocampus by compensatory collateral sprouting from spared cholinergic and noradrenergic afferents, Brain Research, 268 (1983) 27-37. 11 Gage, F.H., Bj6rklund, A., Stenevi, U. and Dunnett, S.B., Functional correlates of compensatory collateral sprouting by aminergic and cholinergic afferents in the hippocampal formation, Brain Research, 268 (1983) 39-47. 12 Jones, B.E. and Moore, R.Y., Ascending projections of the locus coeruleus in the rat. II. Autoradiographic study, Brain Research, 127 (1977) 23-53. 13 Katzman, R., BjOrklund, A., Owman, C., Stenevi, U. and West, K., Evidence for regenerative axon sprouting of central catecholamine neurons in the rat mesencephalon following electrolytic lesions, Brain Research, 25 (1971) 579-596. 14 Keizer, K., Kuypers, H.G.J.M., Huisman, A.M. and Dann, O., Diamidino Yellow dihydrochloride (DY-2HC); a new fluorescent retrograde neuronal tracer which migrates only very slowly out of the cell, Exp. Brain Res., 51 (1983) 179-191. 15 Kiernan, J.A., Hypotheses concerned with axonal regeneration in the mammalian nervous system, Biol. Rev., 54 (1979) 155-197. 16 Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Padt, R.E., Retrograde axonal transport of fluorescent substances in the rat's forebrain, Neurosci. Lett., 6 (1977) 127-135. 17 Mesulam, M.-M., Principles of horseradish peroxidase neurohistochemistry and their applications for tracing neural pathways-axonal transport, enzymes histochemistry and light microscopic analysis. In M.M. Mesulam (Ed.), Tra-
18
19
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22 23
24
25
26
27
cing Neural Connections, John Wiley and Sons, New York, 1982, pp. 1-151. Moore, R.Y., Halanis, A.E. and Jones, B.E., Serotonin neurons of the midbrain raphe: ascending projections, J. Comp. Neurol., 180 (1978) 417-438. Moore, R.Y. and Bloom, F.E., Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems, Annu. Rev. Neurosci., 2 (1979) 113-168. Nygren, L.-G., Fuxe, K., Jonsson, G. and Olson, L., Functional regeneration of 5-hydroxytryptamine nerve terminals in the rat spinal cord following 5,6-dihydroxytryptamine induced degeneration, Brain Research, 78 (1974) 377-394. Paikovits, M. and Jacobowitz, D.M., Topographic atlas of catecholamine and acetylcholinesterase-containing neurons in the rat brain, J. Comp. Neurol., 157 (1974) 29-42. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. Richardson, P.M., Issa, V.M.K. and Aguayo, A.J., Regeneration of long spinal axons in the rat, J. Neurocytol., 13 (1984) 165-182. Salame, C.G. and Dum, R.P., Central nervous system axonal regeneration into sciatic nerve grafts: physiological properties of the grafts and histologic findings in the neuraxis, Exp. Neurol., 90 (1985) 322-340. Stenevi, U. and BjOrklund, A., A pitfall in brain lesion studies: growth of vascular sympathetic axons into the hippocampus after fimbral lesions, Neurosci. Len., 7 (1978) 219-224. Takagi, H., Shiosaka, S., Tohyama, M., Senba, E. and Sakanaka, M., Ascending components of the medial forebrain bundle from the lower brain stem in the rat, with special reference to raphe and catecholamine cell groups. A study by the HRP method, Brain Research, 193 (1980) 315337. Thomas, P.K., Nerve injury. In R, Bellairs and E.G. Gray (Eds.), Essays on the Nervous System, Oxford University Press, London, 1974, pp. 44-70.
Brain Research, 372 (1986) 198-203 Elsevier
BRE 21531
Growth of medial forebrain bundle axons into peripheral nerve grafts in the rat RICHARD P. DUM and CAMILLE G. SALAME Department of Neurosurgery, State University of New York at Upstate Medical Center, Syracuse, NY 13210 (U.S.A.)
(Accepted January 14th, 1986) Key words: regeneration - - medial forebrain bundle - - monoaminergic neuron - - nerve graft
In rats, we intercepted medial forebrain bundle axons just lateral to the hypothalamus with peripheral nerve grafts which terminated extracranially. The neurons which grew into the nerve grafts were labeled with retrogradely transported fluorescent dyes. Catecholamines were labeled with glyoxylic acid histofluorescence. Most nuclei, particularly the raphe complex and locus coeruleus, which project rostraily into the medial forebrain bundle were labeled. Many catecholamine fibers were observed in the graft even after removal of the superior cervical ganglions. Thus, monoaminergic neurons which were located relatively remotely from the implant site exhibited rather selective regrowth into the nerve grafts.
Many different types of central nervous system (CNS) neurons have demonstrated the ability to sprout and elongate into implanted peripheral nerve grafts in rats 1'2's'23'24. The axonal growth into these grafted nerves is usually limited, however, to neurons rather close to the site of the implant. This characteristic of the regrowing neuronal population imposes at least two limitations on the physiological evaluation of these regrowing neurons. First, the diversity of regrowing neurons decreases the probability of a homogeneous effect on potential target neurons. Second, the graft must be implanted so close to the desired neuronal population that their normal morphology and, hence, function may be significantly altered. We have minimized these problems by implanting a nerve graft to intercept monoaminergic axons coursing in the medial forebrain bundle (MFB) 4'5'10'20. This graft location induced a more homogeneous population of neurons to grow without disrupting their normal nuclear morphology. Under aseptic conditions, a small burr hole was made in the skull (1.5 m m lateral, +3.5 mm interaural) of young adult female rats (200 g) which were anesthetized with chloral hydrate (400 mg/kg). A 30-mm piece of autologous sciatic nerve was removed, wrapped around the end of a glass capillary
tube and plunged vertically to the base of the brain. The graft was targeted to interrupt the medial forebrain bundle at the caudal border of the hypothalamus 4. The free end of the graft was led externally for about 15 mm and sutured into the temporalis muscle. Up to one year later, each rat was reanesthetized and the distal end of the graft was mobilized and transected. The cut end was placed on a rubber sheet, surrounded with vaseline and soaked in a tracer substance dissolved in 2% dimethylsulfoxide (DMSO) 8. The tracer substance was one of the following fluorescent dyes: Evans blue (EB), Diamidino yellow (DY), or a mixture of Granular blue (GB) plus horseradish peroxidase (HRP) 3'14'16. The DY treated rat had its superior cervical ganglions removed bilaterally at this time 25. Five days later, GB + H R P treated rats were perfused intracardially with saline, 10% buffered formalin and 10% buffered formalin with 10% glycerin. These brains were blocked and sectioned frozen at 30 ktm. Alternate sections were reacted for H R P by the tetramethyl benzidine method 17. EB- and DY-treated rats were pretreated with Nialamide (400 mg/kg, i.p.) 6 h prior to sacrifice 7. The rats were anesthetized and their brains were removed whole and frozen. Cryostat sections were cut at 8 / t m and reacted by the glyoxylic acid
Correspondence: R.P. Dum, 3118 Weiskotten Hall, 766 Irving Avenue, Syracuse, NY 13210, U.S.A.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
199
A
B250 (.h ._/ .._I t,l
150 I.I.
o ,"r t,l m
:E :m. 50
F t 0
1.8
A
A 3.6
DISTANCE
(ram)
5,4
C
( ! B
4
Imm
Fig. 1. Location and distribution of GB-labeled neurons whose axons grew into a peripheral nerve bridge inserted through the cortex, thalamus and hypothalamus. A: trajectory of graft through thalamus and hypothalamus. Cortex omitted from diagram. B: histogram illustrating the rostrocaudal distribution of labeled neurons. Shaded column indicates location of graft. Arrows correspond to locations of the 3 sections appearing in C in the appropriate left-to-right sequence. C: drawings of 3 sections showing the location of labeled neurons.
method for catecholamine (CA) histofluorescence 9. Every fourth (GB) or sixth (EB, DY) section was examined for fluorescently labeled neurons using a Zeiss microscope with mercury lamp and the 365-nm (GB), 405-nm (CA, DY) and 510-560-nm (EB) filter packs. The labeled neurons were plotted on an X-Y plotter connected to the microscope stage. Nuclear locations and landmarks were established by subsequent counterstaining of the same sections with cresyl violet. Five of the 7 surviving experimental rats exhibited excellent retrograde transport and neuronal labeling. In these cases, the nerve graft intercepted the MFB either at the border between the ventral tegmental area and the substantia nigra or more rostrally in the lateral hypothalamus in the region just medial to the cerebral peduncle and subthalamus (Fig. 1A) 12'18'22. From its termination in the hypothalamic area, the
graft extended dorsally through the thalamus and exited from the cortex. Rostral to the graft, we observed only sparse CA terminal labeling within the ipsilateral hypothalamus. Just caudal to the graft, we found dense accumulations of CA in the area of the MFB on the ipsilateral side as compared to the unoperated side (Fig. 2D). Such dense accumulations of CA have been observed to characterize the delicate, closely packed sprouts of CA fibers which remained for at least 6 months following transection of the MFB 4. These observations provided independent confirmation that the nerve graft did indeed intercept and transect many of the CA axons in the MFB. The location of labeled neurons exhibited a consistent pattern (Fig. 1C, Table I). Nearly all the labeled neurons were caudal to the graft and were located in nuclei which project rostrally into the MFB 26. The
200 raphe complex and locus coeruleus (LC) were always labeled (Table I). The dorsal raphe (DR; Figs. 1C,
2B) contained at least 50% of all labeled cells while the median raphe, raphe pontis and caudal linear nu-
Fig. 2. Photomicrographs of GB-labeled neurons in the dorsal raphe (A, B) and catecholamine fibers in the graft (C) and the MFB (D). A: photomicrograph of GB-labeled neurons in neck of dorsal raphe. Calibration bar, 50/~m. B: photomicrograph of GB-labeled cells primarily in the ipsilateral dorsal raphe. Aqueduct visible at top. Medial longitudinal fasciculivisible on either side of the neck of the DR at bottom. Calibration bar, 200/~m. C: catecholamine-labeled fibers within graft. Bright fibers near top of the photomicrograph delineate the edge of graft. Calibration bar, 25/~m. D: photomicrograph of basal hypothalamus showing dense catecholamine histofluorescence around the MFB just caudal to the graft (left) vs a very sparse amount on the control side (right). The large, dark oval outline of a blood vessel marks the midline.
201 TABLE I Number and location of labeled CNS neurons following application of retrograde tracer substances to the distal end of the peripheral nerve graft implanted in the diencephalon of the rat Nucleus
BBR2 BBR5 BBR6 BBR7 BBRIO
Noradrenergic nuclei A2 0 locus coeruleus 112 parabrachial 4 Raphe nuclei dorsal raphe 549 raphe pontis 35 median raphe 241 caudal linear 36 Tegmental nuclei lateral and dorsal 0 ventral 6 pontine reticular 2 ventral tegmental area 0 interpeduncular 16 pontine central gray 0 hypothalamus 67 pontine and medullary RF 16 zona incerta 0 entopeduncular 0 cortex 0 motor nucleus of 71 0 Total
1084
6 186 0
0 234 0
0 9 0
0 102 11
408 0 30 60
474 36 118 18
146 11 63 17
535 27 63 20
0 0 0
8 0 0
0 1 0
8 0 0
0 0 0 0
6 10 8 0
6 12 0 6
54 0 0 11
0 0 0 0 0
44 0 0 0 132
1 0 0 0 0
3 118 43 1 0
272
995
690
9562
1 Motor nucleus of 7 was probably labeled by fluorescent dye spilled on the terminations of the auriculopalpebralis branch of the 7th nerve and not by growth of motoneurons into the graft within the brain. 2 Total does not include cells in the 7th nucleus.
clei were consistently labeled with significant numbers of neurons. The labeling tended to be ipsilateral although this distinction was somewhat obscured in the midline raphe nuclei (Figs. 1C, 2B). Outside of the MFB nuclei, the hypothalamic, zona incerta and entopeduncular nuclei near the central end of the graft infrequently contained labeled neurons. In particular, only one labeled neuron was ever found adjacent to the graft as it coursed through the cortex and thalamus. Large numbers of neurons were labeled (mean 800 + 330; range 272-1084) in each rat. In contrast to several prior studies 1'8'23'24, most of the regrowing neurons were at least 1.8 mm distant from the cut end of the graft. The typical distribution was bimodal (Fig. 1B), reflecting the large number of neurons in the D R and LC, respectively.
Because most of the labeled neurons arose from nuclei which contain significant monoaminergic populations 7, 3 rat brains were reacted with glyoxylic acid to produce C A histofluorescence in order to directly determine whether CA-containing neurons were growing into the graft. Nearly all of the neurons within LC and A2 which were labeled with EB or D Y were also clearly fluorescent for CA. Even after the removal of both superior cervical ganglions, a diffuse bright cloud of C A fluorescence was observed within the initial segment of the central end of the graft. As the graft continued dorsally through the thalamus, individual C A fibers emerged from the cloud of C A fluorescence, ran parallel to its long axis, and continued within the graft as it exited the cranium (Fig. 2C). However, no fibers were seen within or crossing the epineurium. These observations strongly support the growth of C A axons into the cut central end of the graft. To ensure that the pattern of labeling was not simply due to spillage or leakage of dye onto the cortex, we placed dye directly on rat cortex as a control. Direct application of dye to the cortex produced heavy labeling of cortical neurons at the application site. In contrast, only one labeled cortical neuron was observed in 7 experimental rats, two of which did not exhibit any neuronal labeling. Therefore, any direct spillage of dye onto the cortex would have been easily detected. Our results demonstrated the ability of many neurons which projected within the MFB to grow into peripheral nerve grafts in the rat. Two features of this regrowing neuronal population were of particular interest. First, there was rather selective regrowth of primarily monoaminergic neurons into the nerve graft. Second, most of the regrowing neurons were located relatively remotely from the site of the graft implant. The growth of a specific neuronal population was surprising in view of the fact that most previous reports 1'8'23'24found non-specific ingrowth primarily from neurons adjacent to the graft and a quite precipitous decline in numbers at greater distances. Selective regrowth into nerve grafts may have depended on several factors. First, the type of neuron, as defined by its neurotransmitter, may have been a determining factor 2. The vast majority of our labeled neurons came from nuclei which contain monoaminergic neurotransmitters 7'2~. In particular, nearly all
202 of the neurons in LC which were labeled from the graft were found to contain cateeholamines. Because many of the neurons in the heavily labeled D R and deeper midline raphe nuclei have serotonin as a neurotransmitter TM, some serotonergic neurons were probably among those which projected into the graft. Monoaminergic neurons have demonstrated remarkable abilities to sprout collaterals 1°, to regenerate after chemical lesions 6, to sprout after transection 5'13 and to grow into iris and mitral valve implanted in the brain 4. A number of reasons have been proposed for this remarkable regenerative capacity including unmyelinated fibers 4, their old phylogeny 15 and their ubiquitous, non-specific termination pattern which suggested a neuromodulatory role rather than one for the transmission of specific information 19. A second reason for the rather selective regrowth of MFB neurons into the graft may have been that entry of the regrowing axons was restricted to the cut (open) end of the graft. Such restrictions appeared to apply in regenerating peripheral nerves. Regenerating peripheral axons sprouted into and elongated within the basal lamina tubes containing proliferating Schwann cells 27. Assuming that the same conditions were required for central nervous system axons to elongate within a peripheral nerve graft, the cortical and thalamic neurons located along the course of the nerve graft would have been denied access to open Schwann cell tubes. A related factor was that the epineurium-perineurium sheathing of the nerve graft or glial scar formation in response to the implantation may have provided a sufficient barrier to prevent the entry of CNS axons along the course of the graft. The absence of labeled neurons located along the course of the graft within the cortex and thalamus was not
1 Benfey, M. and Aguayo, A.J., Extensive elongation of axons from rat brain into peripheral nerve grafts, Nature (London), 296 (1982) 150-152. 2 Benfey, M., Bringer, U.R., Vidal-Sanz, M., Bray, G.M. and Aguayo, A.J., Axonal regeneration from GABAergic neurons in the adult rat thalamus, J. Neurocytol., 14 (1985) 279-296. 3 Bentivoglio, M., Kuypers, H.G.J.M., Catsman-Berrevoets, C.E. and Dann, O., Fluorescent retrograde neuronal labeling in rats by means of substances binding specifically to adenine-thymine rich DNA, Neurosci. Lett., 12 (1979) 235-240. 4 Bj6rklund, A. and Stenevi, U., Growth of central catechol-
due to an intrinsic inability of these neurons to regrow. Some neurons within this region have demonstrated the capacity to grow into peripheral nerve grafts 12. A third factor may have been that the central end of the graft always intercepted and presumably interrupted the axons of neurons contributing to the MFB. Although the labeled neurons almost always projected within the MFB, several nuclei which contribute to the MFB were not labeled in our experiments. MFB nuclei which only project into the caudal part of the MFB 26 were unlabeled or sparsely labeled. These nuclei included the following: A1, A2, parabrachial, dorsal and lateral tegmental, and ventral tegmental area. Their axons could have escaped transection. The raphe magnus nucleus projects through the MFB but is located far from the implant site. The absence of labeled cells in this nucleus may reflect the sharply decreasing probability of regrowth which occurs as the distance between the graft and cell body location increases 1's'23'24. Thus, both the presence and the degree of axonal damage may be prerequisites for the initiation of regenerative growth 8,23. The selective regrowth of primarily monoaminergic neurons provides a new opportunity to determine whether these newly growing axons can form functional synapses. It should be possible to implant a graft between the MFB and a denervated hippocampus in order to determine whether monoaminergic reinnervation and behavioral improvement occurs It. Because most of the regrowing neurons are located relatively remotely from the site of the graft implant, only minimal disruption of the normal nuclear morphology and hence functional output should occur.
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