Brain Research, 589 (1992) 115-123 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
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BRES 18022
p75 Nerve growth factor receptor immunoreactivity in the human brainstem and spinal cord Elliott J. M u f s o n , T h o m a s B r a s h e r s - K r u g a n d Jeffrey H. K o r d o w e r Department of Neurological Sciences, Rush Aizheimer's Research Disease Center, Chicago, IL 60612 (USA) (Accepted 31 March 1992)
Key words: Brain; Aged; Neurotrophic; Trophism; Primate; Immunocytochemistry
The distribution of nerve growth factor receptor (NGFR) immunoreactive profiles was investigated in the adult human brainstem and spinal cord using a monoclonal antibody directed against the primate low affinity (p75) NGFR. In the human brainstem, p75NGFR immunoreactive profiles were seen within the mesencephalic and descending nucleus of the trigeminal nerve, the nucleus and tractus solitarius, glossopharyngeal nerve, hypoglossal nucleus, nucleus subtrigeminalis, subnucleus ventralis of the central nucleus of the medulla, nucleus cuneatus and gracilis. At the level of the upper cervical spinal cord, p75NGFR immunoreactive profiles were also seen within the incoming dorsal roots, zone of Lissauer and substantia gelatanosa (lamina II). Virtually no immunoreactivity was associated with cervical spinal cord motor neurons. The demonstration of the p75NGFR in brainstem and spinal cord regions associated with the central transmission of peripheral sensory information suggests that these systems may be influenced by the trophic substance nerve growth factor.
INTRODUCTION
The studies of Levi-Montalcini 24'43 provided evidence that the l l8-amino acid protein nerve growth factor (NGF) acts as atrophic substance modulating the development and maintenance of sympathetic and sensory neurons in the peripheral nervous system. NGF has also been found in the central nervous system (CNS) 48 where its serves a trophic function for basal forebrain cholinergic neurons ~3'ls'2a. Although the precise biochemical mechanism(s) underlying the effects of NGF require further investigation, an important step in NGF-mediated trophism is the binding of the NGF protein to a cell surface receptor located upon NGF responsive cells. The advent of monoclonal antibodies and mRNA probes directed against the low affinity (p75)NGF receptor (NGFR) made it possible to map the location of p75NGFR expressing structures in the brain of many species including rats 4,19,35-37,41,42,49, monkeys2L4°, and h u m a n s L!6'!8'29'3~. Recently, several studies have elucidated the presence
of NGFR protein and message in brain regions within the rodent brainstem and spinal cord 7'~'~'35'36'45'4t'.The p75NGFR may also participate in the formation of receptors for other members of the NGF neurotrophin family 2t'. There are, however, relatively few reports of p75NGF receptor containing profiles in the primate brainstem and spinal cord. For example, NGFR immunoreactivity has been selectively reported within the mesencephalic nucleus of the trigeminal nerve 21 spinal trigeminal nucleus 12 and spinal cord 12'2° in the monkey. However, no systematic studies have assessed the distribution of the p75NGF receptor within the human brainstem and spinal cord. The present investigation used the monoclonal antibody NGFR-5 raised against the primate low affinity NGF receptor to determine the distribution of p75NGF receptor immunoreactivity within the adult human brainstem and spinal cord. We report similar patterns of p75NGFR immunoreactivity in all human cases examined characterized mainly by extensive fiber and neuropil staining with few immunopositive perikarya.
Correspondence: E.J. Mufson, Rush Presbyterian St. Luke's Medical Center, Tech 2000, 2242 W. Harrison St., Suite 200, Chicago, IL 60612, USA. Fax: (1) 312-633-1564.
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Fig. I. Photomicrographs of p75 nerve growth factor receptor (NGFR) immunoreactive brainstem and medullary neurons. A: pseudounipolar perikarya in the mesencephalic nucleus of V. B: multipolar neurons in subnucleus ventralis of the central medulla. C: nucleus subtrigeminalis. Bars: A = 160/zm; C, D = 140/~m.
MATERIALS AND METHODS
Human tissue preparation Brains and spinal cords from 5 male (average age 74.8 years, range 47-90 years) and 4 female (average age 74 years, range 57-98 years) patients without neurologic or psychiatric illness were obtained at autopsy (average postmortem delay 3.8 Ii', range 2-7 h). Each brain was removed from the calvarium and the brainstem was
detached at the pontomesencephalic junction extending from the midbrain through the caudal medulla. In some cases the brainstem was hemisected. The upper cervical spinal cord was severed from the brainstem at the spinal medullary interface. Brain slabs (I cm thick) cut on a plexiglass brain slice apparatus, as well as brainstems and spinal cords, were placed in a solution containing 4% paraformaldehyde (Fisher Scientific Co.) in 0.1 M phosphate buffer (pH 7.4) for 24 h at 4°C. The tissues were then cryoprotected in 10% glycerol plus 2% DMSO in 0.1 M phosphate buffer at 4°C for 2 days followed by
Fig. 2. Coronal sections through the human brainstem immunoreacted with the NGFR5 antibody. A: continuum of NGFR immunoreactivity was seen originating in the glossopharyngeal nerve (n.lX) with fascicles (arrowheads) coursing towards and terminating in the nucleus of the tractus solitarius (thick arrow). Dense NGFR immunoreactivity is also seen within the spinal nucleus of V (Sp.V; arrow) with a decreased staining pattern observed in the descending tract of V (open arrows). B: low power photomicrograph at the level of the olivary nucleus (ion) showing p75NGFR immunostaining in spinal tract of V (SpTtr), solitary nucleus (open arrow) and tract (arrow). C: high magnification photomicrograph of the solitary nucleus (curved arrow) and tract (arrow) seen in B. D: p75NGFR expressing fibers within the trigeminal nerve (Tri.n). Bars: A = 600 ~m; B = 700 ~ m ; C = 500 pm; D = 700 p.m.
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118 immersion in a solution of 20% glycerol plus 2% dimethylsulfoxide (DMSO) -v+ prior to sectioning. Following fixation, the right hemisphere, brainstem and spinal cord were cut into several series of adjacent 40 # m thick sections on a freezing sliding knife microtome in either the coronal or sagittal plane. Sections were collected in 0.1 M phosphate buffer (pH 7.4) and processed immediately for immunohistochemistry or stored in a cryoprotectant solution 3t~'47. Sections from each brain containing the amygdala, hippocampal complex and temporal cortex were also examined for neuritic plaques (NPs) and neurofibrillary tangles (NFTs) with Thioflavin-S and Bielschowsky silver stains ns'`,:'m. Virtually no NPs or NFTs were seen in any of the cases examined. NGFR immunohistochemistry Human sections were processed for NGFR immunohistochemistry using a monoclonal antibody raised against the human low affinity (p75) NGF receptor :x'',~'4° according to a previously described procedure '1'~'~. Briefly, following several rinses in phosphate buffered saline (PBS) tissue sections were incubated for 20 rain in a
Tris-buffered saline solution containing 0.1 M sodium periodate to block endogenous peroxidase staining. After rinses, sections were soaked for ! h in PBS containing 0.3% Triton X-100, 3% normal horse serum and 2% bovine serum albumen (BSA). The primary antibody (NGFR-5; diluted 1:60,000) was applied for 4 h at room temperature with constant agitation and then incubated at 4°C for an additional 48 h. The solvent for the primary antibody contained 0.4% Triton X-100, 1% normal horse serum and !% BSA. The specificity of the NGFR-5 antibody has been described previously 2~'4°. Sections were then incubated in the biotinylated horse antimouse immunoglobulin (1 h at 1:200 dilution; Vector Laboratories)solution. After washes, sections were incubated in the avidin-biotin complex (75 rain at I: 1,000 dilution; 'Elite' Kit, Vector Laboratories). Sections were then rinsed in a 0.2 M sodium acetate-l.0 M imidazole buffer (pH 7.4). The chromogen solution contained 2.5% nickel It sulfate, 0.05% 3', 3'-diaminobenzidine (DAB), and 0.005% H 2 0 2 (pH 7.2). The reaction wa terminated with rinses in the acetate-imidazole buffer. Selected sections were counterstained with thionin to delineate cytoarchitectonic boundaries. Finally, sections were dehydratcd through graded alcohols, cleared in xylene and coverslipped with Permount. Controls consisted of the identical processing of human brainstem and spinal cord sections except that tissue was incubated in the primary antibody solvent. Although control sections generally failed to display specific immunoreaclivity, the possibility that the antibody may react with structurally related proteins cannot be excluded. Thus, the term NGF receptor immunoreactivity in the present investigation refers to p75NGF receptor-'like' immunoreactivity. Structures were identified according to various descriptions of the human brainstem and spinal cord 14"34A4.
RESULTS
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Fig. 3. Coronal section at the level of the lower medulla showing p75NGFR immunostaining within the hypoglossal nucleus (N.Xll), nucleus gracilis (n. gr), nucleus cuneatus (n.cu) and spinal nucleus of V (Sp. V). B: high power photomicrograph of p75NGFR immunoreactivity within the components of the descending nucleus of the trigeminal nerve including the spinal tract of V (SP Ttr), the subnucleus gelatinosus (GL) and the subnucleus magnocellularis (mc). Bars: A, B = 1 mm.
General immunohistochemical characteristics Although most of the human cases can be considered aged, the youngest was 47 years old and did not vary in staining features from other specimens. Thus, we feel that the following descriptions are relevant to the adult population in general and are not specific to aged humans. Immunoreactivity for p75NGFR was seen mainly in fibers and within the lumen of some blood vessels (Fig. 4B) in the brainstem and spinal cord. Sections processed without the primary antibody occasionally displayed faint background staining within the inferior olivary (Fig. 2B) and vestibular nuclei and thus was interpreted as being non-specific. Brainstem NGFR immunoreactive profiles In the mesencephalon, p75NGFR containing neurons were observed within the round perikarya of the mesencephalic nucleus of the trigeminal nerve (Fig. 1A). These cells were large (~ = 54 ~m) and exhibited the classic pseudounipolar morphology of ganglionic neurons. Neurons within the pedunculopontine nucleus (Ch533) were NGFR immunonegative. in the mesencephalon p75NGFR containing fibers were seen within the nerve and tract of the trigeminal nucleus at the level of the inferior olivary nucleus (Fig. 2). The trigeminal nerve contained p75NGFR immunoreactive fascicles which entered the medulla to form discrete bundles within the spinal tract of the
119 trigeminal nerve (Fig. 2A, B and D). At this level, dense p75NGFR immunostaining was also seen within the nucleus and tractus solitarius (Fig. 2A, B and C) which was followed throughout its rostrocadual extent (Fig. 4A). As seen in the coronal plane, p75NGFR immunoreactivity was heavier within the solitary nucleus as compared to the tractus solitarius (Fig. 2C). In addition, p75NGFR immunoreactive fibers were also seen within the glossopharyngeal nerve (N. IX) coursing towards the solitary nucleus (Fig. 2A). A striking collection of dense p75NGFR immunoreactive fibers was observed throughout the rostrocaudal extent of the descending nucleus of the trigeminal tract (Figs. 3 and 4B). A dense crescent shaped band of
p75NGFR immunoreactivity was seen within the laterally situated subnucleus gelatinosus portion of the spinal trigeminal nucleus (Fig. 3A and B) with a diminution of this staining pattern within its inner magnocellular region (Fig. 3B). NGFR staining was also seen in the hypoglossal nucleus (n.XII) (Fig. 3A). Dense p75NGFR immunostaining was found within the neuropil of the nucleus gracilis and cuneatus (Fig. 3A). This staining pattern was limited to p75NGFR immunoreactive fibers. In contrast, only a few immunoreactive fibers were seen coursing within their associated fasciculi. Virtually no perikarya were p75NGFR positive in these nuclei, p75NGFR immunoreactive neurons were found within the nucleus
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Fig. 4. Sagittal sections of the human brainstem demonstrating p75NGFR immunostaining within (A) the solitary nucleus (n. Sol.) and (B) the descending spinal nucleus of V and its spinal cord homologue, the substantia gelatanosa. Arrow indicates the location of an NGFR positive blood vessel. Bars: A, B = ! mm.
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121 subtrigeminalis and subnucleus ventralis of the central nucleus of the lower medulla (Fig. 1B and C).
Spinal cord p 75NGFR immunoreactit,ity Examination of the spinal cord was limited to the upper cervical region. At this level p75NGFR immunoreactive fibers were seen within the dorsal root (Fig. 5A) and Lissauer's tract (Fig. 4B and C). A dense p75NGFR fiber network was also observed within the substantia gelatinosa (lamina II) (Fig. 5B and C), the spinal cord homologue of the descending spinal nucleus of V. Light to moderate p75NGFR immunoreactive profiles extended through laminae Ill-IV. At the cervical spinal cord level, the large anterior horn cells were p75NGFR immunonegative. DISCUSSION
The present investigation demonstrates p75NGFR immunoreactive perikarya, fibers and blood vessels within the human brainstem and spinal cord corresponding to the pattern seen in the rat 35'36, cat ~2 and monkey t2.22. However, unlike cholinergic basal forebrain neurons which express the p75NGF receptor, cholinergic cells located within the pedunculopontine nucleus 33 of the brainstem were p75NGFR im• 16 '27 '4t~. The origin of p75NGFR immunonegatwe munoreactivity seen within the continuum of staining extending from the spinal trigeminal complex to the substantia gelatanosa has yet to be conclusively determined. Since CNS structures which project to these regions do not display p75NGFR perikarya ''J, it is likely that the fiber staining in this nucleus is derived from the peripheral nervous system. Spinal primary sensory 24 and trigeminal 5° ganglia which are responsive to NGF and contain the NGF protein and mRNA, are likely candidates as a source of these fibers. p75NGFR staining was also observed within the nucleus cuneatus and gracilis but not in their associated fasciculi. The persistence of p75NGFR staining in these nuclei in the adult human is in contrast to previous reports demonstrating that p75NGFR is only transiently expressed in these nuclei in the developing rodent '~. Since we were unable to visualize p75NGFR positive cell bodies within these nuclei, the source of the receptor staining remains to be determined. How-
ever, these findings suggest that NGF/p75NGFR trophic systems play a role in the modulation of peripherally derived tactile and kinesthetic information. The presence of p75NGFR staining in the glossopharyngeal nerve (n.IX) and the solitary nucleus and tract suggests that NGF may modulate other visceral and sensory information. The p75NGFR containing fibers seen within the n.IX which were traced to the nucleus of the tractus solitarius probably originate in the cell bodies of the petrosal ganglion 44. These are general visceral afferent fibers conveying tactile, pain and thermal sense from the posterior portior.~ of the tongue, tonsil and Eustachian tube. More numerous are the special visceral afferent fibers from the taste buds of the posterior third of the tongue which travel in the glossopharyngeal nerve. Interestingly, after entering the medulla, these fibers contribute to the upper portion of the tractus solitarius and terminate in the rostral aspect of the gustatory nucleus. The gustatory portion of the nucleus solitarius stains intensely for the low affinity NGF receptor. Moreover, the caudal tractus solitarius and nucleus also express intense p75NGFR immunoreactivity suggesting that NGF is involved in autonomic innervation arising from the vagus nerve. Taken together these finding suggest that the trophic substance NGF may be involved in a complex anatomical circuitry underlying sensory and autonomic functions. The distribution of p75NGFR immunoreactivity seen in the spinal nucleus of the trigeminal nerve and its spinal homologue the substantia gelatanosa is similar to that described for various neuropeptides. For.example, galanin, substance P and calcitonin gene-related peptide (CGRP) immunoreactivity are seen in these structures in cat ~2, monkey ~2 and humans 2"~. In fact, the dense continuum of p75NGFR fibers within the substantia gelatanosa displays a striking overlap with the distribution of these neuropeptides. Interestingly, a subset of dorsal root ganglia express galanin and CGRF immunoreactivity ~. It would interesting to determine the degree to which p75NGFR and these neuropeptides colocalize within dorsal root ganglia and fibers within the brainstem and spinal cord, since not all p75NGFR fibers found in these brainstem structures simultaneously express CGRP in the monkey 12. The functional significance of the overlap of the NGFR and
Fig. 5. Coronal sections through the upper cervical spinal cord immunoreacted for p75NGFR. A: p75NGFR immunoreactive fibers of a dorsal root (DR) entering the cervical spinal cord. B: low power photomicrograph of an upper cervical spinal section immunoreacted for p75NGFR and counterstained for Nissl substance showing the location of the tract of Lissauer (small arrow) and the substantia gelatanosa (open arrow). Note the location of the spinal motor neurons (arrow) which are stained darkly for Nissl substance but were NGFR immunonegative. C: high power photomicrograph showing the tract of Lissauer (Lt) and substantia gelantanosus (S. Gel). Bars: 600 p.m.
122 various neuropeptides remains to be determined. However, the presence of NGFR and these neuropeptides in similar termination fields suggests an interactive modulatory role for these wateins in primary sensory processing a. The presence of the p75NGFR in brainstem and spinal cord systems underlying the central transmission of peripheral sensory information suggests that these systems may be influenced by the trophic substance NGF. It should be noted that the p75NGFR40 antibody, recognizes the low affinity form of the NGF receptor. Therefore, it cannot unequivocally be determined that the NGF receptor visualizes an active site of the NGF regulated trophism since this action takes place through the high affinity receptor site. It is important to note, however, the presence of the low affinity site appears to be necessary for trophic effects to o c c u r 17.
The entry of molecular biology into the field of neurotrophic substances has recently led to the discovery that NGF is a member of a family of neurotrophins including brain derived neurotrophic factor (BDNF) and NT-3 which exhibit functional and structural homologies to N(}F 4'-~'2~'38'43. Recent investigations indicate that in addition to NGF, developing dorsal root ganglia respond to BDNF in vitro ~. BDNF mRNA has also been found in a subset of rodent dorsal root ganglia cells t~. Since no synapses are located upon perikarya of the dorsal root ganglia, NGF-like neurotrophins produced in these neurons may be transported to the central nervous system and released from terminal fields in order to act as a neurotrophic factor ~. it is also possible that the NGF-like trophins produced in dorsal root ganglia cells regulate neuronal activity by an autocrine mechanism as proposed for other populations of neurons containing the p75NGFR :-~.3~.32. The functional importance of the expression of the NGF family of neurotrophins in the brainstem and spinal cord remains to be elucidated. Finally, it has become apparent that the N G F / NGFR complex probably plays a crucial role in peripheral nerve survival. For example, axotomy of the adult rat sciatic nerve induces expression of both the protein and mRNA for NGF in Schwann cells proximal to injury 42. Similarly, axotomy induces the expression of the low affinity NGFR in the hypoglossal nucleus in rats:. These experimental findings suggest that the expression of the receptor for NGF mediates atrophic function for regenerating peripheral sensory neuronal systems. Interestingly, we observed p75NGFR immunoreactivity within the hypoglossal nucleus in humans without peripheral nerve damage. Thus, it is possible that the NGF/p75NGFR complex may partic-
ipate in peripheral nervous systems regenerative events in humans. Indeed, NGF may be a potential therapeutic agent for the treatment of peripheral nerve trauma. Acknowledgements. The authors thank R. Casanova and L. Sue for histological assistance. This research is supported by AG 09466 (J.H.K.), AG 10668 (E.J.M.), American Health Assistance Foundation and Illinois Public Health Service Grant (E.J.M.).
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115
BRES 18022
p75 Nerve growth factor receptor immunoreactivity in the human brainstem and spinal cord Elliott J. M u f s o n , T h o m a s B r a s h e r s - K r u g a n d Jeffrey H. K o r d o w e r Department of Neurological Sciences, Rush Aizheimer's Research Disease Center, Chicago, IL 60612 (USA) (Accepted 31 March 1992)
Key words: Brain; Aged; Neurotrophic; Trophism; Primate; Immunocytochemistry
The distribution of nerve growth factor receptor (NGFR) immunoreactive profiles was investigated in the adult human brainstem and spinal cord using a monoclonal antibody directed against the primate low affinity (p75) NGFR. In the human brainstem, p75NGFR immunoreactive profiles were seen within the mesencephalic and descending nucleus of the trigeminal nerve, the nucleus and tractus solitarius, glossopharyngeal nerve, hypoglossal nucleus, nucleus subtrigeminalis, subnucleus ventralis of the central nucleus of the medulla, nucleus cuneatus and gracilis. At the level of the upper cervical spinal cord, p75NGFR immunoreactive profiles were also seen within the incoming dorsal roots, zone of Lissauer and substantia gelatanosa (lamina II). Virtually no immunoreactivity was associated with cervical spinal cord motor neurons. The demonstration of the p75NGFR in brainstem and spinal cord regions associated with the central transmission of peripheral sensory information suggests that these systems may be influenced by the trophic substance nerve growth factor.
INTRODUCTION
The studies of Levi-Montalcini 24'43 provided evidence that the l l8-amino acid protein nerve growth factor (NGF) acts as atrophic substance modulating the development and maintenance of sympathetic and sensory neurons in the peripheral nervous system. NGF has also been found in the central nervous system (CNS) 48 where its serves a trophic function for basal forebrain cholinergic neurons ~3'ls'2a. Although the precise biochemical mechanism(s) underlying the effects of NGF require further investigation, an important step in NGF-mediated trophism is the binding of the NGF protein to a cell surface receptor located upon NGF responsive cells. The advent of monoclonal antibodies and mRNA probes directed against the low affinity (p75)NGF receptor (NGFR) made it possible to map the location of p75NGFR expressing structures in the brain of many species including rats 4,19,35-37,41,42,49, monkeys2L4°, and h u m a n s L!6'!8'29'3~. Recently, several studies have elucidated the presence
of NGFR protein and message in brain regions within the rodent brainstem and spinal cord 7'~'~'35'36'45'4t'.The p75NGFR may also participate in the formation of receptors for other members of the NGF neurotrophin family 2t'. There are, however, relatively few reports of p75NGF receptor containing profiles in the primate brainstem and spinal cord. For example, NGFR immunoreactivity has been selectively reported within the mesencephalic nucleus of the trigeminal nerve 21 spinal trigeminal nucleus 12 and spinal cord 12'2° in the monkey. However, no systematic studies have assessed the distribution of the p75NGF receptor within the human brainstem and spinal cord. The present investigation used the monoclonal antibody NGFR-5 raised against the primate low affinity NGF receptor to determine the distribution of p75NGF receptor immunoreactivity within the adult human brainstem and spinal cord. We report similar patterns of p75NGFR immunoreactivity in all human cases examined characterized mainly by extensive fiber and neuropil staining with few immunopositive perikarya.
Correspondence: E.J. Mufson, Rush Presbyterian St. Luke's Medical Center, Tech 2000, 2242 W. Harrison St., Suite 200, Chicago, IL 60612, USA. Fax: (1) 312-633-1564.
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Fig. I. Photomicrographs of p75 nerve growth factor receptor (NGFR) immunoreactive brainstem and medullary neurons. A: pseudounipolar perikarya in the mesencephalic nucleus of V. B: multipolar neurons in subnucleus ventralis of the central medulla. C: nucleus subtrigeminalis. Bars: A = 160/zm; C, D = 140/~m.
MATERIALS AND METHODS
Human tissue preparation Brains and spinal cords from 5 male (average age 74.8 years, range 47-90 years) and 4 female (average age 74 years, range 57-98 years) patients without neurologic or psychiatric illness were obtained at autopsy (average postmortem delay 3.8 Ii', range 2-7 h). Each brain was removed from the calvarium and the brainstem was
detached at the pontomesencephalic junction extending from the midbrain through the caudal medulla. In some cases the brainstem was hemisected. The upper cervical spinal cord was severed from the brainstem at the spinal medullary interface. Brain slabs (I cm thick) cut on a plexiglass brain slice apparatus, as well as brainstems and spinal cords, were placed in a solution containing 4% paraformaldehyde (Fisher Scientific Co.) in 0.1 M phosphate buffer (pH 7.4) for 24 h at 4°C. The tissues were then cryoprotected in 10% glycerol plus 2% DMSO in 0.1 M phosphate buffer at 4°C for 2 days followed by
Fig. 2. Coronal sections through the human brainstem immunoreacted with the NGFR5 antibody. A: continuum of NGFR immunoreactivity was seen originating in the glossopharyngeal nerve (n.lX) with fascicles (arrowheads) coursing towards and terminating in the nucleus of the tractus solitarius (thick arrow). Dense NGFR immunoreactivity is also seen within the spinal nucleus of V (Sp.V; arrow) with a decreased staining pattern observed in the descending tract of V (open arrows). B: low power photomicrograph at the level of the olivary nucleus (ion) showing p75NGFR immunostaining in spinal tract of V (SpTtr), solitary nucleus (open arrow) and tract (arrow). C: high magnification photomicrograph of the solitary nucleus (curved arrow) and tract (arrow) seen in B. D: p75NGFR expressing fibers within the trigeminal nerve (Tri.n). Bars: A = 600 ~m; B = 700 ~ m ; C = 500 pm; D = 700 p.m.
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118 immersion in a solution of 20% glycerol plus 2% dimethylsulfoxide (DMSO) -v+ prior to sectioning. Following fixation, the right hemisphere, brainstem and spinal cord were cut into several series of adjacent 40 # m thick sections on a freezing sliding knife microtome in either the coronal or sagittal plane. Sections were collected in 0.1 M phosphate buffer (pH 7.4) and processed immediately for immunohistochemistry or stored in a cryoprotectant solution 3t~'47. Sections from each brain containing the amygdala, hippocampal complex and temporal cortex were also examined for neuritic plaques (NPs) and neurofibrillary tangles (NFTs) with Thioflavin-S and Bielschowsky silver stains ns'`,:'m. Virtually no NPs or NFTs were seen in any of the cases examined. NGFR immunohistochemistry Human sections were processed for NGFR immunohistochemistry using a monoclonal antibody raised against the human low affinity (p75) NGF receptor :x'',~'4° according to a previously described procedure '1'~'~. Briefly, following several rinses in phosphate buffered saline (PBS) tissue sections were incubated for 20 rain in a
Tris-buffered saline solution containing 0.1 M sodium periodate to block endogenous peroxidase staining. After rinses, sections were soaked for ! h in PBS containing 0.3% Triton X-100, 3% normal horse serum and 2% bovine serum albumen (BSA). The primary antibody (NGFR-5; diluted 1:60,000) was applied for 4 h at room temperature with constant agitation and then incubated at 4°C for an additional 48 h. The solvent for the primary antibody contained 0.4% Triton X-100, 1% normal horse serum and !% BSA. The specificity of the NGFR-5 antibody has been described previously 2~'4°. Sections were then incubated in the biotinylated horse antimouse immunoglobulin (1 h at 1:200 dilution; Vector Laboratories)solution. After washes, sections were incubated in the avidin-biotin complex (75 rain at I: 1,000 dilution; 'Elite' Kit, Vector Laboratories). Sections were then rinsed in a 0.2 M sodium acetate-l.0 M imidazole buffer (pH 7.4). The chromogen solution contained 2.5% nickel It sulfate, 0.05% 3', 3'-diaminobenzidine (DAB), and 0.005% H 2 0 2 (pH 7.2). The reaction wa terminated with rinses in the acetate-imidazole buffer. Selected sections were counterstained with thionin to delineate cytoarchitectonic boundaries. Finally, sections were dehydratcd through graded alcohols, cleared in xylene and coverslipped with Permount. Controls consisted of the identical processing of human brainstem and spinal cord sections except that tissue was incubated in the primary antibody solvent. Although control sections generally failed to display specific immunoreaclivity, the possibility that the antibody may react with structurally related proteins cannot be excluded. Thus, the term NGF receptor immunoreactivity in the present investigation refers to p75NGF receptor-'like' immunoreactivity. Structures were identified according to various descriptions of the human brainstem and spinal cord 14"34A4.
RESULTS
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Fig. 3. Coronal section at the level of the lower medulla showing p75NGFR immunostaining within the hypoglossal nucleus (N.Xll), nucleus gracilis (n. gr), nucleus cuneatus (n.cu) and spinal nucleus of V (Sp. V). B: high power photomicrograph of p75NGFR immunoreactivity within the components of the descending nucleus of the trigeminal nerve including the spinal tract of V (SP Ttr), the subnucleus gelatinosus (GL) and the subnucleus magnocellularis (mc). Bars: A, B = 1 mm.
General immunohistochemical characteristics Although most of the human cases can be considered aged, the youngest was 47 years old and did not vary in staining features from other specimens. Thus, we feel that the following descriptions are relevant to the adult population in general and are not specific to aged humans. Immunoreactivity for p75NGFR was seen mainly in fibers and within the lumen of some blood vessels (Fig. 4B) in the brainstem and spinal cord. Sections processed without the primary antibody occasionally displayed faint background staining within the inferior olivary (Fig. 2B) and vestibular nuclei and thus was interpreted as being non-specific. Brainstem NGFR immunoreactive profiles In the mesencephalon, p75NGFR containing neurons were observed within the round perikarya of the mesencephalic nucleus of the trigeminal nerve (Fig. 1A). These cells were large (~ = 54 ~m) and exhibited the classic pseudounipolar morphology of ganglionic neurons. Neurons within the pedunculopontine nucleus (Ch533) were NGFR immunonegative. in the mesencephalon p75NGFR containing fibers were seen within the nerve and tract of the trigeminal nucleus at the level of the inferior olivary nucleus (Fig. 2). The trigeminal nerve contained p75NGFR immunoreactive fascicles which entered the medulla to form discrete bundles within the spinal tract of the
119 trigeminal nerve (Fig. 2A, B and D). At this level, dense p75NGFR immunostaining was also seen within the nucleus and tractus solitarius (Fig. 2A, B and C) which was followed throughout its rostrocadual extent (Fig. 4A). As seen in the coronal plane, p75NGFR immunoreactivity was heavier within the solitary nucleus as compared to the tractus solitarius (Fig. 2C). In addition, p75NGFR immunoreactive fibers were also seen within the glossopharyngeal nerve (N. IX) coursing towards the solitary nucleus (Fig. 2A). A striking collection of dense p75NGFR immunoreactive fibers was observed throughout the rostrocaudal extent of the descending nucleus of the trigeminal tract (Figs. 3 and 4B). A dense crescent shaped band of
p75NGFR immunoreactivity was seen within the laterally situated subnucleus gelatinosus portion of the spinal trigeminal nucleus (Fig. 3A and B) with a diminution of this staining pattern within its inner magnocellular region (Fig. 3B). NGFR staining was also seen in the hypoglossal nucleus (n.XII) (Fig. 3A). Dense p75NGFR immunostaining was found within the neuropil of the nucleus gracilis and cuneatus (Fig. 3A). This staining pattern was limited to p75NGFR immunoreactive fibers. In contrast, only a few immunoreactive fibers were seen coursing within their associated fasciculi. Virtually no perikarya were p75NGFR positive in these nuclei, p75NGFR immunoreactive neurons were found within the nucleus
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Fig. 4. Sagittal sections of the human brainstem demonstrating p75NGFR immunostaining within (A) the solitary nucleus (n. Sol.) and (B) the descending spinal nucleus of V and its spinal cord homologue, the substantia gelatanosa. Arrow indicates the location of an NGFR positive blood vessel. Bars: A, B = ! mm.
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121 subtrigeminalis and subnucleus ventralis of the central nucleus of the lower medulla (Fig. 1B and C).
Spinal cord p 75NGFR immunoreactit,ity Examination of the spinal cord was limited to the upper cervical region. At this level p75NGFR immunoreactive fibers were seen within the dorsal root (Fig. 5A) and Lissauer's tract (Fig. 4B and C). A dense p75NGFR fiber network was also observed within the substantia gelatinosa (lamina II) (Fig. 5B and C), the spinal cord homologue of the descending spinal nucleus of V. Light to moderate p75NGFR immunoreactive profiles extended through laminae Ill-IV. At the cervical spinal cord level, the large anterior horn cells were p75NGFR immunonegative. DISCUSSION
The present investigation demonstrates p75NGFR immunoreactive perikarya, fibers and blood vessels within the human brainstem and spinal cord corresponding to the pattern seen in the rat 35'36, cat ~2 and monkey t2.22. However, unlike cholinergic basal forebrain neurons which express the p75NGF receptor, cholinergic cells located within the pedunculopontine nucleus 33 of the brainstem were p75NGFR im• 16 '27 '4t~. The origin of p75NGFR immunonegatwe munoreactivity seen within the continuum of staining extending from the spinal trigeminal complex to the substantia gelatanosa has yet to be conclusively determined. Since CNS structures which project to these regions do not display p75NGFR perikarya ''J, it is likely that the fiber staining in this nucleus is derived from the peripheral nervous system. Spinal primary sensory 24 and trigeminal 5° ganglia which are responsive to NGF and contain the NGF protein and mRNA, are likely candidates as a source of these fibers. p75NGFR staining was also observed within the nucleus cuneatus and gracilis but not in their associated fasciculi. The persistence of p75NGFR staining in these nuclei in the adult human is in contrast to previous reports demonstrating that p75NGFR is only transiently expressed in these nuclei in the developing rodent '~. Since we were unable to visualize p75NGFR positive cell bodies within these nuclei, the source of the receptor staining remains to be determined. How-
ever, these findings suggest that NGF/p75NGFR trophic systems play a role in the modulation of peripherally derived tactile and kinesthetic information. The presence of p75NGFR staining in the glossopharyngeal nerve (n.IX) and the solitary nucleus and tract suggests that NGF may modulate other visceral and sensory information. The p75NGFR containing fibers seen within the n.IX which were traced to the nucleus of the tractus solitarius probably originate in the cell bodies of the petrosal ganglion 44. These are general visceral afferent fibers conveying tactile, pain and thermal sense from the posterior portior.~ of the tongue, tonsil and Eustachian tube. More numerous are the special visceral afferent fibers from the taste buds of the posterior third of the tongue which travel in the glossopharyngeal nerve. Interestingly, after entering the medulla, these fibers contribute to the upper portion of the tractus solitarius and terminate in the rostral aspect of the gustatory nucleus. The gustatory portion of the nucleus solitarius stains intensely for the low affinity NGF receptor. Moreover, the caudal tractus solitarius and nucleus also express intense p75NGFR immunoreactivity suggesting that NGF is involved in autonomic innervation arising from the vagus nerve. Taken together these finding suggest that the trophic substance NGF may be involved in a complex anatomical circuitry underlying sensory and autonomic functions. The distribution of p75NGFR immunoreactivity seen in the spinal nucleus of the trigeminal nerve and its spinal homologue the substantia gelatanosa is similar to that described for various neuropeptides. For.example, galanin, substance P and calcitonin gene-related peptide (CGRP) immunoreactivity are seen in these structures in cat ~2, monkey ~2 and humans 2"~. In fact, the dense continuum of p75NGFR fibers within the substantia gelatanosa displays a striking overlap with the distribution of these neuropeptides. Interestingly, a subset of dorsal root ganglia express galanin and CGRF immunoreactivity ~. It would interesting to determine the degree to which p75NGFR and these neuropeptides colocalize within dorsal root ganglia and fibers within the brainstem and spinal cord, since not all p75NGFR fibers found in these brainstem structures simultaneously express CGRP in the monkey 12. The functional significance of the overlap of the NGFR and
Fig. 5. Coronal sections through the upper cervical spinal cord immunoreacted for p75NGFR. A: p75NGFR immunoreactive fibers of a dorsal root (DR) entering the cervical spinal cord. B: low power photomicrograph of an upper cervical spinal section immunoreacted for p75NGFR and counterstained for Nissl substance showing the location of the tract of Lissauer (small arrow) and the substantia gelatanosa (open arrow). Note the location of the spinal motor neurons (arrow) which are stained darkly for Nissl substance but were NGFR immunonegative. C: high power photomicrograph showing the tract of Lissauer (Lt) and substantia gelantanosus (S. Gel). Bars: 600 p.m.
122 various neuropeptides remains to be determined. However, the presence of NGFR and these neuropeptides in similar termination fields suggests an interactive modulatory role for these wateins in primary sensory processing a. The presence of the p75NGFR in brainstem and spinal cord systems underlying the central transmission of peripheral sensory information suggests that these systems may be influenced by the trophic substance NGF. It should be noted that the p75NGFR40 antibody, recognizes the low affinity form of the NGF receptor. Therefore, it cannot unequivocally be determined that the NGF receptor visualizes an active site of the NGF regulated trophism since this action takes place through the high affinity receptor site. It is important to note, however, the presence of the low affinity site appears to be necessary for trophic effects to o c c u r 17.
The entry of molecular biology into the field of neurotrophic substances has recently led to the discovery that NGF is a member of a family of neurotrophins including brain derived neurotrophic factor (BDNF) and NT-3 which exhibit functional and structural homologies to N(}F 4'-~'2~'38'43. Recent investigations indicate that in addition to NGF, developing dorsal root ganglia respond to BDNF in vitro ~. BDNF mRNA has also been found in a subset of rodent dorsal root ganglia cells t~. Since no synapses are located upon perikarya of the dorsal root ganglia, NGF-like neurotrophins produced in these neurons may be transported to the central nervous system and released from terminal fields in order to act as a neurotrophic factor ~. it is also possible that the NGF-like trophins produced in dorsal root ganglia cells regulate neuronal activity by an autocrine mechanism as proposed for other populations of neurons containing the p75NGFR :-~.3~.32. The functional importance of the expression of the NGF family of neurotrophins in the brainstem and spinal cord remains to be elucidated. Finally, it has become apparent that the N G F / NGFR complex probably plays a crucial role in peripheral nerve survival. For example, axotomy of the adult rat sciatic nerve induces expression of both the protein and mRNA for NGF in Schwann cells proximal to injury 42. Similarly, axotomy induces the expression of the low affinity NGFR in the hypoglossal nucleus in rats:. These experimental findings suggest that the expression of the receptor for NGF mediates atrophic function for regenerating peripheral sensory neuronal systems. Interestingly, we observed p75NGFR immunoreactivity within the hypoglossal nucleus in humans without peripheral nerve damage. Thus, it is possible that the NGF/p75NGFR complex may partic-
ipate in peripheral nervous systems regenerative events in humans. Indeed, NGF may be a potential therapeutic agent for the treatment of peripheral nerve trauma. Acknowledgements. The authors thank R. Casanova and L. Sue for histological assistance. This research is supported by AG 09466 (J.H.K.), AG 10668 (E.J.M.), American Health Assistance Foundation and Illinois Public Health Service Grant (E.J.M.).
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