0306-4522/9 I $3.00+ 0.00 Pergamon Press plc
NeuroscienceVol. 40, No. 3, pp. 713-724, 1991 Printed in Great Britain
NADPH
0 1991 IBRO
DIAPHORASE HISTOCHEMISTRY HUMAN HYPOTHALAMUS
OF THE
T. SANGRUCHI and N. W. KOWALL* Department of Neuropathology
and Neurology Service, Massachusetts General Hospital, Boston, MA 02114, U.S.A.
Abstract-The morphology and distribution of NADPH diaphorase reactive neurons was studied in the normal human hypothalamus. Reactive neurons were divided into three categories on the basis of perikaryal size. Small neurons (8-20 pm) were oval or fusiform, and pale staining. Intermediate neurons (2&30 lrn) were fusiform, triangular or pyramidal with a wide range of staining intensity. Large neurons
(> 30 pm) were triangular or pyramidal with moderate to dark staining. Reactive neurons were found in four major regions: medial preoptic, ventromedial, lateral, and perifomical. Scattered positive neurons were found in several other hypothalamic areas. Reactive fibers were present in the supraoptic decussation, medial forebrain bundle, and stria medullaris thalami. The localization of NADPH diaphorase neurons in hypothalamic nuclei affected by Alzheimer’s disease and other degenerative disorders suggests that further studies of this neuronal subset are warranted.
The hypothalamus encompasses a heterogeneous collection of nuclei that serve to integrate endocrine and autonomic activity and mediate complex behavioral phenomena.26*29 The general organization of the hypothalamus is uniform in all mammalian species but important phylogenetic differences exist.2%30v33 Abnormalities of the human hypothalamus have been found in neurodegenerative diseases such as Alzheimer’s and Huntington’s diseases that may be responsible for abnormal sleep patterns and cachexia.i0*i9 In Huntington’s disease, a subset of striatal neurons containing somatostatin, neuropeptide Y, and the enzyme reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase are preserved relative to other neuronal types.3,5 In Alzheimer’s disease, cortical somatostatin immunoreactivity is depleted out of proportion to other peptides, thus suggesting that somatostatin neurons, which often contain NADPH diaphorase activity, are disproportionately sensitive to degeneration.” In the present study we examined the normal anatomy of NADPH diaphorase reactive neurons in the human hypothalamus to define features unique to human hypothalamus and provide a baseline for studies of neurodegenerative disorders such as Alzheimer’s and Huntington’s diseases. EXPERIMENTAL PROCEDURES Seven normal brains were obtained from neurologically normal patients less than 24 h after death (age range 1690 years, mean 58 years). Coronal blocks extending from the optic chiasm to the mammillary bodies were taken and fixed at 4°C for 2448 h in periodate-lysine-paraformaldehyde fixative23 followed by cryoprotection in 20% glycerol and
2% dimethylsulfoxide for at least 48 h. Serial sections were cut in either the coronal or horizontal plane on a sliding microtome at 50pm and collected as sets stored at 4°C in 0.1 M phosphate buffer, pH 7.3. Incubation medium for NADPH diaphorase contained 1 mM NADPH, 0.8 mM Nitroblue Tetrazolium, 8 mM monosodium malate, and 0.8% Triton X-100 in 0.1 M phosphate buffer, pH 8.0.” Sections were incubated in the dark at 37°C and monitored by intermittent microscopic examination. One serial set from each case was counterstained with Cresyl Violet. Representative sections from six levels of hypothalamus were charted with a projection microscope. Perikaryal dimensions were measured with an eyepiece graticule. Anatomical definitions are after Nauta and Haymaker. RESULTS Cytology
Three categories of neurons were defined on the basis of neuronal size and staining intensity. Small neurons (8-20 pm) generally stained faintly. Cell bodies were oval, fusiform or triangular with one to three long primary dendrites. Secondary dendrites were sparse. They were predominantly found in the preoptic and ventromedial regions (Fig. 1). Intermediate neurons (2&30 pm) varied in staining intensity. Cell bodies were triangular or fusiform with two to three primary dendrites of variable length. They were found in lateral hypothalamus, ventromedial, and tuberomammillary region (Fig. 2). Large neurons (>30 pm) usually stained medium to dark. Large fusiform or triangular neurons had two to three primary dendrites with longer secondary dendrites. Large neurons formed a dense cluster in the lateral hypothalamus (Fig. 3). Distribution
*To whom correspondence should be addressed. Abbreoiarion: NADPH, reduced nicotinamide adenine dinucleotide phosphate.
of NADPH
diaphorase neurons andfibers
Four major clusters of NADPH diaphorase reactive neurons were defined. Scattered reactive neurons 713
714
T. SANGRUCHI and N. W.
Fig. I. Photomicrographs area; (B) lateral preoptic
KOWAL.I
of NADPH diaphorase reactive neurons in hypothalamus: (A) medial preoptic area; (C) ventromedial area; (D) lateral hypothalamic area. Scale bar = 20 pm.
were found elsewhere in hypothalamus. A number of adjacent structures also had prominent NADPH diaphorase reactive neurons and fibers. Medial preoptic group. This small cluster, composed of monomorphic small neurons, was located in the medial preoptic region. The center of the cluster (cell density 610/400 x field) was just inferior to the anterior commissure. A small number of short, pale staining fibers was seen in the neuropil. Laterally, they mixed with intermediate neurons and merged with the lateral group. Ventrally, NADPH neurons were fewer in number and absent in the suprachiasmatic and periventricular regions (Fig. 4). Ventromedialgroup. This larger group was centered in the ventromedial nucleus and extended into the dorsomedial nucleus. Maximum cell density was 25-30/400x field. Small and intermediate neurons were present. Neuropil stained more darkly than in the preoptic group and contained a number of short fibers (Fig. 5).
Lateralgroup. The largest group was situated in the lateral hypothalamus. It is distinguished by its content of darkly stained small, intermediate or large neurons embedded in strongly reactive parallel fiber bundles. From the preoptic level, scattered neurons merged with neurons of the medial preoptic group and extended in continuity with NADPH diaphorase neurons in the substantia innominata. Highest cell density (15-20/400 x field) was between the lateral border of the column of the fornix and medial border of the pallidum and the internal capsule. Caudally, darkly stained neurons were sandwiched between the internal capsule and the subthalamic nucleus. Strongly NADPH diaphorase reactive fibers from this region were in continuity with fibers from the substantia innominata. Some fibers traversed the pallidum to the internal capsule and could be traced along the medial surface of the caudate and thalamus to the lateral habenular nucleus. A small number of intermediate and large neurons were scattered in the
715
NADPH diaphorase in human h~thaiamus
distribution patterns of NADPH diaphorase reactive cells in the hypothalamus. Strongly reactive fibers were found in the dorsal aspect of the optic chiasm and tract, most clearly in horizontal sections. A few positive fibers were found in the periphery of the anterior ~~issure. Intermediate neurons were rarely seen within the substance of the commissure. A small number of large neurons were moderately reactive and a few fibers were seen in the neuropil of the external segment of the globus pallidus. Densely reactive varicose fibers formed a ~~~e~ntial plexus su~ounding the internal segment of the globus pallidus and interpolated between the internal and external segments. Fibers could be followed ventromedially along the ansa lenticularis. Moderately reactive large neurons were found in the medial portion and in circumferential fibers. Parallel fiber bundles were seen in the stria medullaris thalami and lateral habenula. Neurons were not reactive.
DISCUSSION
Fig. 2. Photomicrographs of NADPH diaphorase reactive neurons in hypothalamus: (A) lateral hypothalamic area medial to globus pallidus; (B) perifornical area; (C) ventromedial area. Scale bar = 20 pm.
lateral aspect of the mammillothalamic tract (Figs 6 and 7). Perifornical group. This group was defined separately because neurons and fibers tended to wrap around the fornix. As the fornix descended to the mammilla~ body, the density of inte~ediate neurons increased to as much as 70/400x field and formed a ring-like pattern. A few neurons were embedded within the fornix. Inferiorly, adjacent to the mammillary body, these neurons intermingled with reactive neurons in the tuberomammillary region (Fig. 8). Other ~~pot~aia~ic nuclei and adjacent structures.
Small numbers of NADPH diaphorase found in several hypothalamic nuclei form specific groups (Table 1, Fig. 9). show projection microscope charts of
neurons were that did not Figures 10-15 representative
The functional importance of NADPH diaphorase in neurons where it is concentrated is unknown. We have previously speculated that this enzyme may function to synthesize reduced NADP as classically defined or to oxidize an as yet undefined substrate chemically related to quinones.4*‘6’6 Of interest in this regard is the recent description of the amino acid sequence of adenylyl cyclase, the enzyme responsible for cyclic adenosine monophosphate synthesis. These results indicate that this brain enzyme has several transmembrane domains suggesting that it may function to transport cyclic adenosine monophosphate from cell~.‘~ Characte~zation of the NADPH diaphorase enzyme may provide similar unexpected insight into its functional properties. Despite the uncertainty regarding the function of NADPH diaphorase, it is clear that NADPH diaphorase histochemistry defines a consistent and unique pattern of reactive neurons and fibers in all cases. Their distribution pattern is generally similar to that previously reported in cat and monkey brain, but differences exist.g In both cat and monkey prominent reactive neurons were found in rostra1 anterior hypothalamus and paraventricular nucleus where we found only scattered neurons. Moderate numbers of reactive neurons were reported in dorso- and ventromedial nuclei of cat and monkey where we found a similarly prominent group. In both species NADPH diaphorase neurons are prominent caudally in the posterior hypothalamus, supramammillary decussations, and tu~romammilla~ nucleus. In human, positive cells form a more dense, compact group. Reactive neurons in perifornical and intrafornical nuclei were defined that do not exist in lower animals. Strongly reactive neurons and fibers were present in lateral hypothalamus in all species, including man.
716
T. SANGRU~HI and N’. W.
KOWALI
Fig. 3. Comparison of hypothalamic NADPH diaphorase neurons with reactive neurons from other sites: (A) lateral hypothalamus; (B) posterior hypothalamus; (C) substantia innominata; (D) caudate nucleus; (E) globus pallidus externa; (F) globus patlidus interna. Scale bar = 20 pm.
NADPH diaphorase in human hypothalamus
Fig. 4. (A) Cluster of NADPH diaphorase positive neurons in preoptic hypothalamus (ac, anteriorcommisure; pr, preoptic periventricular area; 1, lateral preoptic area). Scale bar = 200 pm. (B) Higher power shows small neurons. Scale bar = 20 pm.
717
718
T. SANGKUCHIand N. W. KOWALI.
Fig. 5. in veni
1Infundibular level of hy~thalamus showing group of NADPH diaphorase neureas ten &red medialnucleus (pv, periventricular area). Scale bar = 200 pm. (B) Higher pov Jer shows mi.xture of small and intermediate size neurons. Scale bar = 20,~m.
NADPH diaphorase in human hypothalamus
Fig. 6. NADPH diaphorase neurons in lateral hypothalamic area. (A) Infundibular level (f, fornix). Scale bar = 500 pm. (B, C) neurons lateral to the fornix. Scale bar = 50 pm. (D) Reactive neurons medial to the internal capsule in the lateral hypothalamus. Scale bar = 50 pm.
719
Suprachiasmatic, supraoptic, infundibular, and mammillary nuclei were non-reactive in all studies. The cluster of large NADPH diaphorase neurons in the lateral hypothalamus continues into the substantia innominata4 embedded in positive fibers of the medial forebrain bundle that run to the amygdala, lateral hypothalamus and the substantia innominata. NADPH diaphorase positive fibers can also be traced along the stria medullaris thalami into the lateral habenular nucleus. They may constitute part of the lateral olfactohabenular tract or amygdalohypothalamic connections originating in the lateral hypothalamus.7~26Prominent reactive fibers in the dorsal optic chiasm can be traced into the ventral pallidum and lateral midbrain.i6 The source of these fibers may be the peripeduncular nucleus which contributes to the ventral supraoptic decussation of Gudden.24 In primate brain the peripeduncular nucleus, nucleus basalis and amygdala are linked by a common projection which crosses in the ventral supraoptic decussation.12 Dark staining neurons are prominent in the medial segment of the internal pallidum and internal medullary lamina. Despite the uniformity of morphological cell types between external and internal pallidum and substantia nigra reticulata, only the internal segment contains reactive neurons which are not otherwise distinctive morphologically from other pallidal neurons. ’ The hypothalamus is especially rich in peptides and other neurotransmitters. Peptides are especially prominent in the infundibular, supraoptic and paraventricular nuclei which do not contain significant numbers of NADPH diaphorase neurons. There is some correspondence, however, between defined neurotransmitter systems and the distribution of NADPH diaphorase neurons. Atria1 natriuretic peptide is found in the medial preoptic region.36 The ventromedial ,region contains substance P, somatostatin, and enkephalin.6~“~21 Lateral hypothalamus contains acetylcholinesterase and choline acetyltransferase reactive neurons, calcitonin generelated peptide and galanin.‘3~28,3’~32Galanin and acetylcholinesterase are also found in the perifomical nucleus. It is not known if cholinergic neurons in the hypothalamus contain NADPH diaphorase. In the striatum these substances exist in separate neuronal populations whereas in the nucleus tegmenti dorsalis lateralis they are co-localized.34 Acetylcholinesterase neurons are also found in the infundibular and supramammillary nucleus.28 Choline acetyltransferase positive neurons are found mainly in the infundibular nucleus with scattered cells in the lateral hypothalamus. 32Substance P neurons are present in the bed nucleus of the stria terminalis, medial amygdala, medial habenular and infundibular nucleus and project via the stria medullaris, terminalis and fasciculus retroflexuq2’ all of which contain NADPH diaphorase reactive fibers. Somatostatin and neuropeptide Y are often co-localized with NADPH
Fig. 7. NADPH diaphorase neurons at the level of the mammillothalamic large size neurons lateral to the mammillothalamic tract.
tract. (A, B) Intermediate Scale bar = 2Opm.
and
Fig. 8. NADPH diaphorase neurons at the rostra1 margin of the mammillary body. (A) Reactive neurons and fibers form a shell-like structure around the fornix (0. Scale bar = 500 pm. Fibers (B) and intermediate size reactive neurons (C) are present. Scale bar = 50 /tcm (B); 20 ,um (C). 770
721
NADPH diaphorase in human hypothalamus
Table 1. Summary of characteristics of NADPH diaphorase neuronal clusters in hypothalamus* Group
Shape
Size
Intensity
Density
Anterior Paraventricular Tuberomammillary Posterior
Oval Fusiform Fusiformltriangular Fusiform/triangular
Small Intermediate Intermediate Intermediate/large
Pale Dark Pale (medium) Medium dark
+ + +++ + +
*Reactive neurons were not found in periventricular, suprachiasmatic, supraoptic, infundibular, tuberolateral and mammillary nuclei. Size: small, 8-20 pm; intermediate, 20-30 pm; large, 3@40 pm. Density: + , O-2/400 x field; + + , l-3/400 x field; + + t , 335/400 x field.
Fig. 9. NADPH diaphorase neurons in other hypothalamic regions: (A) paraventricular nucleus; (B) tuberomammillary (TM) nucleus; (C) suprammammillary nucleus. Scale bar = 30 pm. Dark appearing lipofuscin-positive neurons in the tuberolateral (TL) nucleus are not NADPH diaphorase positive (D). Positive neurons and fibers surround the nucleus. Scale bar = 50lm.
722
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l
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,° °:
.
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Fig. 12. Chart showing distribution of prominent reactive neurons and fibers in the lateral hypothalamus (L). Scattered positive neurons were found in paraventricular (PV), dorsomedial (DM), and tuberomammillary (TM) nuclei. Infundibular (IF) and tuberolateral (TL) nuclei were negative (F, fornix). Scale bar = 2 mm.
f
Fig. 10. Chart of NADPH diaphorase neurons and fibers at the level of the optic chiasm (OC). Larger neurons congregate in lateral preoptic area and extend into the substantia innominata (SI). Reactive fibers were present in the medial forebrain bundle lateral to the fornix (F) and in the supraoptic decussation. Scale bar = 2 ram. Fig. 13. Chart at the maximum extent of the mammillary body (M) shows prominent perifornical group. Scale bar = 2 ram.
l
F
Fig. 11. Chart of NADPH diaphorase neurons in ventromedial nucleus (VM) and lateral hypothalamic area (L). The paraventrieular nucleus (PV) was non-reactive (OT, optic tract). Scale bar = 2 ram.
diaphorase in the forebrain, l~ Neuropeptide Y and somatostatin neurons are primarily found in supraoptic, paraventricular and infundibular nuclei which are N A D P H diaphorase negative. 27 In Alzheimer's disease, senile plaques and neurofibrillary tangles are found in the hypothalamus in 92% of cases. 22 They are most prominent in the lateral hypothalamus, tuberomammillary, dorsomedial and posterior hypothalamus} ° In the brainstem, Mufson and Mash ~5 showed that N A D P H
;
":~'~#~,::"". ~ e ~ ' ~ .
i ~ . ~
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Fig. 14. Chart at level of the fornix (F) merging with the mammillary body (M). Perifornical neurons are noted. Lateral hypothalamic fibers in continuity from the ventral pallidum and amygdala. Scale bar = 2 mm,
diaphorase reactive neurons develop neurofibrillary tangles. Neurofibrillary change has also been described in the hypothalamus in other diseases such as G u a m a n i a n Parkinson's dementia and postencephalitic Parkinsonism. s Hypothalamic lesions have also been described in Huntington's disease brain. Neuronal shrinkage
NADPH diaphorase in human hypothalamus
//
123
and hyperchromasia were found in the tuberoand mamlateral, supraoptic, paraventricular millary nuclei. 35 In the striatum and possibly the cerebral cortex NADPH diaphorase neurons are resistant to damage in Huntington’s disease.’ It is not known if NADPH diaphorase neurons are spared in the human hypothalamus but it is clear that this unique subset of hypothalamic neurons should be studied in neurodegenerative disorders.
/
Acknowledgements-We
Fig. 15. Chart at the level of the mammillothalamic tract (MT) shows prominent cluster lateral to the tract and in posterior hypothalamus. Scale bar = 2 mm.
thank Michelle Forrestall, Steve Conley and Larry Cherkas for photographic skills and Bernard J. Quigley Jr for technical assistance. Thanks to Dr E. P. Richardson for helpful discussions and review of the material. T. Sangruchi is a visiting fellow supported by the Siriraj Foundation, Siriraj Hospital, Mahidol University, Bangkok, Thailand. Supported by NIH grants AGO5134 andNS25588.
REFERENCES 1. Carpenter M. B. and Sutin J. (1983) The corpus striatum. In Human Neuroanaromy, pp. 584586. Williams and Wilkins, Maryland. 2. Christ J. F. (1969) Derivation and boundaries of the hypothalamus with atlas of hypothalamic grisea. In The Hypothalamus (ed. Haymaker W.), pp. 13-59. Thomas, Springfield. 3. Dawbam D., DeQuidt M. E. and Emson P. C. (1985) Survival of basal ganglia neuropeptide Y-somatostatin neurons in Huntington’s disease. Brain Res. 340, 251-260. 4. Ellison D. W., Kowall N. W. and Martin J. B. (1987) A neuronal subset characterized by NADPH diaphorase activity in human substantia innominata. J. camp: Neurol. 260, 233-247. 5. Ferrante R. J., Kowall N. W., Beal M. F., Richardson E. P. Jr, Bird E. D. and Martin J. B. (1985) Selective sparing of a class of striatal neurons in Huntington’s disease. Science 230, 561-563. 6. Haber S. and Elde R. (1982) The distribution of enkephalin immunoreactive fibers and terminals in the monkey central nervous system. Neuroscience 7, 1049-1095. I. Herkenham M. and Nauta W. J. H. (1977) Afferent connections of the habenular nuclei in the rat: a horseradish peroxidase study with a note on the fiber of passage problem. J. camp. Neurol. 173, 123-145. 8. Hirano A. and Zimmerman H. M. (1962) Alzheimer’s neurofibrillary changes: a topographic study. Archs Neural., Chicago 7, 227-242.
9. Hu H., Jayashree K. R. and Jayaraman A. (1987) The distribution of NADPH diaphorase reactive neurons in the hypothalamus: a study in cats and rhesus monkeys. Sot. Neurosci. Absrr. 13, 1165. 10. Ishii T. (1966) Distribution of Alzheimer’s neurofibrillary changes in the brainstem and hypothalamus of senile dementia. Acta Neuropath. 6, 181-187. 11. Johansson O., Hokfelt T. and Elde R. P. (1984) Immunohistochemical distribution of somatostatin like immunoreactivity in the central nervous system of the adult cat. Neuroscience 13, 265-339. 12. Jones E. G., Burton H., Saper C. B. and Swanson L. W. (1976) Midbrain, diencephalic and cortical relationship of the basal nucleus of Meynert and associated structures in primate. J. camp. Neurol. 167, 385420. 13. Kawai Y., Takami K., Shiosaka S., Emson P. C. and Hillyard C. J. (1985) Topographic localization of calcitonin gene-related peptide in the rat brain. Neuroscience 15, 747-763. 14. Kowall N. W., Ferrante R. J., Beal M. F., Richardson E. P. Jr, Sofroniew M. V., Cuello A. C. and Martin J. B. (1987) Neuropeptide Y, somatostatin, and NADPH diaphorase in the human striatum: a combined immunocytochemical and enzyme histochemical study. Neuroscience 20, 817-828. 15. Kowall N. W. and Beal M. F. (1988) Cortical somatostatin, neuropeptide Y and NADPH diaphorase neurons: normal anatomy and alterations in Alzheimer’s disease. Ann. Neurol. 23, 105-l 14. 16. Kowall N. W. and Mueller M. P. (1988) Morphology and distribution of NADPH diaphorase neurons in human brainstem. Neuroscience 26, 645-654. staining neurons 17. Kowall N. W., Beal M. F., Ferrante R. J. and Martin J. B. (1985) Topography of NADPHdiaphorase in rat striatum. Neurosci. Lptf. 59, 6146. 18. Krupinski J., Coussen F., Bakalyar H. A., Tang W.-J., Feinstein P. G., Orth K., Slaughter C., Reed R. R. and Gilman A. G. (1989) Adenylyl cyclase amino acid sequence: possible channel- or transporter-like structure. Science 244, 1558-1564. 19. Langston J. W. and Forno L. S. (1978) The hypothalamus in Parkinson’s disease. Ann. Neural. 3, 129-133. 20. Lee D. 0. F. and Markesbery W. R. (1986) The hypothalamus in senile dementia of the Alzheimer type. J. Neuropath. exp. Neurol. 45, 341.
21. Mai J. K., Stephens P. H., Hopf A. and Cue110A. C. (1986) Substance Pin the human brain. Neuroscience 17,709-739. 22. McDuff T. and Sumi S. M. (1985) Subcortical degeneration in Alzheimer’s disease. Neurology 35, 123-126. fixative a new fixative for immunoelectron 23 McLean I. W. and Nakane P. K. (1974) Periodatelysine-paraformaldehyde microscopy. J. Histochem. Cytochem. 22, 1077-1083.
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SANGRUCHI and
N. W. KOWALL
24. MettIer F. A. (1968) Anatomy of the basal ganglia. In Handbook of Neurology (eds Vinken P. J. and Bruyri G. W.). Vol. 6, pp. l-55. Wiley and Sons, New York. 25. Mufson E. J. and Mash D. C. (1987) Pathology in brainstem cholinergic neurons in Alzheimer’s disease. Sot.. ,~errro~i. Abstr. 13, 1461. 26. Nauta W. J. H. and Haymaker W. (1969) Hypothalamic nuclei and fiber connections, In The Hypotholumur (ed. Haymaker W.), pp. 136-207. Thomas, Springfield. 27. O’Donohue T. L., Chronwall B. M., Pruss R. M., Mezey E., Kiss J. Z., Eiden L. E., Massari V. J., Hotchkiss A. J., Crowley W. R. and Zukowska-Grojec Z. (1985) Neuropeptide Y and peptide YY neuronal and endocrine systems. Peptides 6, 755-768.
28. Parent A., Poirier L. J., Boucher R. and Butcher L. L. (1977) Morphological characteristics of acetylcholinesterase containing neurons in the central nervous system of DFP treated monkey. J. Neural. Sci. 32, 9-28. 29. Reichlin S., Baldessarini R. T. and Martin J. B. (1978) Nonendocrine disease and disorders of the hypothalamus. In The Hypothalamus (eds Plum F. and Uitert R. V.), pp. 415-172. Raven Press, New York. 30. Rose J. (1939) The cell structure of the mammillary body. J. An&. 74, 91-115. 31. Skofitsch G. and Jacobowitz D. M. (1985) Immunohistochemical mapping of galanin-like neurons in the rat central nervous system. Peptides 6, 509-546. 32. Tago H., McGeer P. L., Bruce G. and Hersh L. B. (1987) Distribution of choline acetyltransferase containing neurons of the hypothalamus. Brain Res. 415, 49-62. 33. Valenstein E. S. and Nauta W. J. H. (1959) A comparison of the distribution of the fornix system in rat, guinea pig, cat and monkey. J. camp. Neural. 113, 337-363. 34. Vincent S. R., Satoh K., Armstrong D. M. and Fibiger H. C. (1983) NADPH diaphorase a selective histochemical marker for the cholinergic neurons of the pontine reticular formation. Neurosci. Left. 43, 31-36. 35. Wahren W. (1952) The changes of hypothalamic nuclei in schizophrenia. Proc. Int. Gong. Neuroparh. 3, 660-673. 36. Zamir N., Skofitsch G., Eskay R. L. and Jacobowitz D. M. (1986) Distribution of immunoreactive atria1 natriuretic peptide in the central nervous system of the rat. Brain Rex 365, 105-l 11. (Accepted 20 August 1990)
NeuroscienceVol. 40, No. 3, pp. 713-724, 1991 Printed in Great Britain
NADPH
0 1991 IBRO
DIAPHORASE HISTOCHEMISTRY HUMAN HYPOTHALAMUS
OF THE
T. SANGRUCHI and N. W. KOWALL* Department of Neuropathology
and Neurology Service, Massachusetts General Hospital, Boston, MA 02114, U.S.A.
Abstract-The morphology and distribution of NADPH diaphorase reactive neurons was studied in the normal human hypothalamus. Reactive neurons were divided into three categories on the basis of perikaryal size. Small neurons (8-20 pm) were oval or fusiform, and pale staining. Intermediate neurons (2&30 lrn) were fusiform, triangular or pyramidal with a wide range of staining intensity. Large neurons
(> 30 pm) were triangular or pyramidal with moderate to dark staining. Reactive neurons were found in four major regions: medial preoptic, ventromedial, lateral, and perifomical. Scattered positive neurons were found in several other hypothalamic areas. Reactive fibers were present in the supraoptic decussation, medial forebrain bundle, and stria medullaris thalami. The localization of NADPH diaphorase neurons in hypothalamic nuclei affected by Alzheimer’s disease and other degenerative disorders suggests that further studies of this neuronal subset are warranted.
The hypothalamus encompasses a heterogeneous collection of nuclei that serve to integrate endocrine and autonomic activity and mediate complex behavioral phenomena.26*29 The general organization of the hypothalamus is uniform in all mammalian species but important phylogenetic differences exist.2%30v33 Abnormalities of the human hypothalamus have been found in neurodegenerative diseases such as Alzheimer’s and Huntington’s diseases that may be responsible for abnormal sleep patterns and cachexia.i0*i9 In Huntington’s disease, a subset of striatal neurons containing somatostatin, neuropeptide Y, and the enzyme reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase are preserved relative to other neuronal types.3,5 In Alzheimer’s disease, cortical somatostatin immunoreactivity is depleted out of proportion to other peptides, thus suggesting that somatostatin neurons, which often contain NADPH diaphorase activity, are disproportionately sensitive to degeneration.” In the present study we examined the normal anatomy of NADPH diaphorase reactive neurons in the human hypothalamus to define features unique to human hypothalamus and provide a baseline for studies of neurodegenerative disorders such as Alzheimer’s and Huntington’s diseases. EXPERIMENTAL PROCEDURES Seven normal brains were obtained from neurologically normal patients less than 24 h after death (age range 1690 years, mean 58 years). Coronal blocks extending from the optic chiasm to the mammillary bodies were taken and fixed at 4°C for 2448 h in periodate-lysine-paraformaldehyde fixative23 followed by cryoprotection in 20% glycerol and
2% dimethylsulfoxide for at least 48 h. Serial sections were cut in either the coronal or horizontal plane on a sliding microtome at 50pm and collected as sets stored at 4°C in 0.1 M phosphate buffer, pH 7.3. Incubation medium for NADPH diaphorase contained 1 mM NADPH, 0.8 mM Nitroblue Tetrazolium, 8 mM monosodium malate, and 0.8% Triton X-100 in 0.1 M phosphate buffer, pH 8.0.” Sections were incubated in the dark at 37°C and monitored by intermittent microscopic examination. One serial set from each case was counterstained with Cresyl Violet. Representative sections from six levels of hypothalamus were charted with a projection microscope. Perikaryal dimensions were measured with an eyepiece graticule. Anatomical definitions are after Nauta and Haymaker. RESULTS Cytology
Three categories of neurons were defined on the basis of neuronal size and staining intensity. Small neurons (8-20 pm) generally stained faintly. Cell bodies were oval, fusiform or triangular with one to three long primary dendrites. Secondary dendrites were sparse. They were predominantly found in the preoptic and ventromedial regions (Fig. 1). Intermediate neurons (2&30 pm) varied in staining intensity. Cell bodies were triangular or fusiform with two to three primary dendrites of variable length. They were found in lateral hypothalamus, ventromedial, and tuberomammillary region (Fig. 2). Large neurons (>30 pm) usually stained medium to dark. Large fusiform or triangular neurons had two to three primary dendrites with longer secondary dendrites. Large neurons formed a dense cluster in the lateral hypothalamus (Fig. 3). Distribution
*To whom correspondence should be addressed. Abbreoiarion: NADPH, reduced nicotinamide adenine dinucleotide phosphate.
of NADPH
diaphorase neurons andfibers
Four major clusters of NADPH diaphorase reactive neurons were defined. Scattered reactive neurons 713
714
T. SANGRUCHI and N. W.
Fig. I. Photomicrographs area; (B) lateral preoptic
KOWAL.I
of NADPH diaphorase reactive neurons in hypothalamus: (A) medial preoptic area; (C) ventromedial area; (D) lateral hypothalamic area. Scale bar = 20 pm.
were found elsewhere in hypothalamus. A number of adjacent structures also had prominent NADPH diaphorase reactive neurons and fibers. Medial preoptic group. This small cluster, composed of monomorphic small neurons, was located in the medial preoptic region. The center of the cluster (cell density 610/400 x field) was just inferior to the anterior commissure. A small number of short, pale staining fibers was seen in the neuropil. Laterally, they mixed with intermediate neurons and merged with the lateral group. Ventrally, NADPH neurons were fewer in number and absent in the suprachiasmatic and periventricular regions (Fig. 4). Ventromedialgroup. This larger group was centered in the ventromedial nucleus and extended into the dorsomedial nucleus. Maximum cell density was 25-30/400x field. Small and intermediate neurons were present. Neuropil stained more darkly than in the preoptic group and contained a number of short fibers (Fig. 5).
Lateralgroup. The largest group was situated in the lateral hypothalamus. It is distinguished by its content of darkly stained small, intermediate or large neurons embedded in strongly reactive parallel fiber bundles. From the preoptic level, scattered neurons merged with neurons of the medial preoptic group and extended in continuity with NADPH diaphorase neurons in the substantia innominata. Highest cell density (15-20/400 x field) was between the lateral border of the column of the fornix and medial border of the pallidum and the internal capsule. Caudally, darkly stained neurons were sandwiched between the internal capsule and the subthalamic nucleus. Strongly NADPH diaphorase reactive fibers from this region were in continuity with fibers from the substantia innominata. Some fibers traversed the pallidum to the internal capsule and could be traced along the medial surface of the caudate and thalamus to the lateral habenular nucleus. A small number of intermediate and large neurons were scattered in the
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NADPH diaphorase in human h~thaiamus
distribution patterns of NADPH diaphorase reactive cells in the hypothalamus. Strongly reactive fibers were found in the dorsal aspect of the optic chiasm and tract, most clearly in horizontal sections. A few positive fibers were found in the periphery of the anterior ~~issure. Intermediate neurons were rarely seen within the substance of the commissure. A small number of large neurons were moderately reactive and a few fibers were seen in the neuropil of the external segment of the globus pallidus. Densely reactive varicose fibers formed a ~~~e~ntial plexus su~ounding the internal segment of the globus pallidus and interpolated between the internal and external segments. Fibers could be followed ventromedially along the ansa lenticularis. Moderately reactive large neurons were found in the medial portion and in circumferential fibers. Parallel fiber bundles were seen in the stria medullaris thalami and lateral habenula. Neurons were not reactive.
DISCUSSION
Fig. 2. Photomicrographs of NADPH diaphorase reactive neurons in hypothalamus: (A) lateral hypothalamic area medial to globus pallidus; (B) perifornical area; (C) ventromedial area. Scale bar = 20 pm.
lateral aspect of the mammillothalamic tract (Figs 6 and 7). Perifornical group. This group was defined separately because neurons and fibers tended to wrap around the fornix. As the fornix descended to the mammilla~ body, the density of inte~ediate neurons increased to as much as 70/400x field and formed a ring-like pattern. A few neurons were embedded within the fornix. Inferiorly, adjacent to the mammillary body, these neurons intermingled with reactive neurons in the tuberomammillary region (Fig. 8). Other ~~pot~aia~ic nuclei and adjacent structures.
Small numbers of NADPH diaphorase found in several hypothalamic nuclei form specific groups (Table 1, Fig. 9). show projection microscope charts of
neurons were that did not Figures 10-15 representative
The functional importance of NADPH diaphorase in neurons where it is concentrated is unknown. We have previously speculated that this enzyme may function to synthesize reduced NADP as classically defined or to oxidize an as yet undefined substrate chemically related to quinones.4*‘6’6 Of interest in this regard is the recent description of the amino acid sequence of adenylyl cyclase, the enzyme responsible for cyclic adenosine monophosphate synthesis. These results indicate that this brain enzyme has several transmembrane domains suggesting that it may function to transport cyclic adenosine monophosphate from cell~.‘~ Characte~zation of the NADPH diaphorase enzyme may provide similar unexpected insight into its functional properties. Despite the uncertainty regarding the function of NADPH diaphorase, it is clear that NADPH diaphorase histochemistry defines a consistent and unique pattern of reactive neurons and fibers in all cases. Their distribution pattern is generally similar to that previously reported in cat and monkey brain, but differences exist.g In both cat and monkey prominent reactive neurons were found in rostra1 anterior hypothalamus and paraventricular nucleus where we found only scattered neurons. Moderate numbers of reactive neurons were reported in dorso- and ventromedial nuclei of cat and monkey where we found a similarly prominent group. In both species NADPH diaphorase neurons are prominent caudally in the posterior hypothalamus, supramammillary decussations, and tu~romammilla~ nucleus. In human, positive cells form a more dense, compact group. Reactive neurons in perifornical and intrafornical nuclei were defined that do not exist in lower animals. Strongly reactive neurons and fibers were present in lateral hypothalamus in all species, including man.
716
T. SANGRU~HI and N’. W.
KOWALI
Fig. 3. Comparison of hypothalamic NADPH diaphorase neurons with reactive neurons from other sites: (A) lateral hypothalamus; (B) posterior hypothalamus; (C) substantia innominata; (D) caudate nucleus; (E) globus pallidus externa; (F) globus patlidus interna. Scale bar = 20 pm.
NADPH diaphorase in human hypothalamus
Fig. 4. (A) Cluster of NADPH diaphorase positive neurons in preoptic hypothalamus (ac, anteriorcommisure; pr, preoptic periventricular area; 1, lateral preoptic area). Scale bar = 200 pm. (B) Higher power shows small neurons. Scale bar = 20 pm.
717
718
T. SANGKUCHIand N. W. KOWALI.
Fig. 5. in veni
1Infundibular level of hy~thalamus showing group of NADPH diaphorase neureas ten &red medialnucleus (pv, periventricular area). Scale bar = 200 pm. (B) Higher pov Jer shows mi.xture of small and intermediate size neurons. Scale bar = 20,~m.
NADPH diaphorase in human hypothalamus
Fig. 6. NADPH diaphorase neurons in lateral hypothalamic area. (A) Infundibular level (f, fornix). Scale bar = 500 pm. (B, C) neurons lateral to the fornix. Scale bar = 50 pm. (D) Reactive neurons medial to the internal capsule in the lateral hypothalamus. Scale bar = 50 pm.
719
Suprachiasmatic, supraoptic, infundibular, and mammillary nuclei were non-reactive in all studies. The cluster of large NADPH diaphorase neurons in the lateral hypothalamus continues into the substantia innominata4 embedded in positive fibers of the medial forebrain bundle that run to the amygdala, lateral hypothalamus and the substantia innominata. NADPH diaphorase positive fibers can also be traced along the stria medullaris thalami into the lateral habenular nucleus. They may constitute part of the lateral olfactohabenular tract or amygdalohypothalamic connections originating in the lateral hypothalamus.7~26Prominent reactive fibers in the dorsal optic chiasm can be traced into the ventral pallidum and lateral midbrain.i6 The source of these fibers may be the peripeduncular nucleus which contributes to the ventral supraoptic decussation of Gudden.24 In primate brain the peripeduncular nucleus, nucleus basalis and amygdala are linked by a common projection which crosses in the ventral supraoptic decussation.12 Dark staining neurons are prominent in the medial segment of the internal pallidum and internal medullary lamina. Despite the uniformity of morphological cell types between external and internal pallidum and substantia nigra reticulata, only the internal segment contains reactive neurons which are not otherwise distinctive morphologically from other pallidal neurons. ’ The hypothalamus is especially rich in peptides and other neurotransmitters. Peptides are especially prominent in the infundibular, supraoptic and paraventricular nuclei which do not contain significant numbers of NADPH diaphorase neurons. There is some correspondence, however, between defined neurotransmitter systems and the distribution of NADPH diaphorase neurons. Atria1 natriuretic peptide is found in the medial preoptic region.36 The ventromedial ,region contains substance P, somatostatin, and enkephalin.6~“~21 Lateral hypothalamus contains acetylcholinesterase and choline acetyltransferase reactive neurons, calcitonin generelated peptide and galanin.‘3~28,3’~32Galanin and acetylcholinesterase are also found in the perifomical nucleus. It is not known if cholinergic neurons in the hypothalamus contain NADPH diaphorase. In the striatum these substances exist in separate neuronal populations whereas in the nucleus tegmenti dorsalis lateralis they are co-localized.34 Acetylcholinesterase neurons are also found in the infundibular and supramammillary nucleus.28 Choline acetyltransferase positive neurons are found mainly in the infundibular nucleus with scattered cells in the lateral hypothalamus. 32Substance P neurons are present in the bed nucleus of the stria terminalis, medial amygdala, medial habenular and infundibular nucleus and project via the stria medullaris, terminalis and fasciculus retroflexuq2’ all of which contain NADPH diaphorase reactive fibers. Somatostatin and neuropeptide Y are often co-localized with NADPH
Fig. 7. NADPH diaphorase neurons at the level of the mammillothalamic large size neurons lateral to the mammillothalamic tract.
tract. (A, B) Intermediate Scale bar = 2Opm.
and
Fig. 8. NADPH diaphorase neurons at the rostra1 margin of the mammillary body. (A) Reactive neurons and fibers form a shell-like structure around the fornix (0. Scale bar = 500 pm. Fibers (B) and intermediate size reactive neurons (C) are present. Scale bar = 50 /tcm (B); 20 ,um (C). 770
721
NADPH diaphorase in human hypothalamus
Table 1. Summary of characteristics of NADPH diaphorase neuronal clusters in hypothalamus* Group
Shape
Size
Intensity
Density
Anterior Paraventricular Tuberomammillary Posterior
Oval Fusiform Fusiformltriangular Fusiform/triangular
Small Intermediate Intermediate Intermediate/large
Pale Dark Pale (medium) Medium dark
+ + +++ + +
*Reactive neurons were not found in periventricular, suprachiasmatic, supraoptic, infundibular, tuberolateral and mammillary nuclei. Size: small, 8-20 pm; intermediate, 20-30 pm; large, 3@40 pm. Density: + , O-2/400 x field; + + , l-3/400 x field; + + t , 335/400 x field.
Fig. 9. NADPH diaphorase neurons in other hypothalamic regions: (A) paraventricular nucleus; (B) tuberomammillary (TM) nucleus; (C) suprammammillary nucleus. Scale bar = 30 pm. Dark appearing lipofuscin-positive neurons in the tuberolateral (TL) nucleus are not NADPH diaphorase positive (D). Positive neurons and fibers surround the nucleus. Scale bar = 50lm.
722
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Fig. 12. Chart showing distribution of prominent reactive neurons and fibers in the lateral hypothalamus (L). Scattered positive neurons were found in paraventricular (PV), dorsomedial (DM), and tuberomammillary (TM) nuclei. Infundibular (IF) and tuberolateral (TL) nuclei were negative (F, fornix). Scale bar = 2 mm.
f
Fig. 10. Chart of NADPH diaphorase neurons and fibers at the level of the optic chiasm (OC). Larger neurons congregate in lateral preoptic area and extend into the substantia innominata (SI). Reactive fibers were present in the medial forebrain bundle lateral to the fornix (F) and in the supraoptic decussation. Scale bar = 2 ram. Fig. 13. Chart at the maximum extent of the mammillary body (M) shows prominent perifornical group. Scale bar = 2 ram.
l
F
Fig. 11. Chart of NADPH diaphorase neurons in ventromedial nucleus (VM) and lateral hypothalamic area (L). The paraventrieular nucleus (PV) was non-reactive (OT, optic tract). Scale bar = 2 ram.
diaphorase in the forebrain, l~ Neuropeptide Y and somatostatin neurons are primarily found in supraoptic, paraventricular and infundibular nuclei which are N A D P H diaphorase negative. 27 In Alzheimer's disease, senile plaques and neurofibrillary tangles are found in the hypothalamus in 92% of cases. 22 They are most prominent in the lateral hypothalamus, tuberomammillary, dorsomedial and posterior hypothalamus} ° In the brainstem, Mufson and Mash ~5 showed that N A D P H
;
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Fig. 14. Chart at level of the fornix (F) merging with the mammillary body (M). Perifornical neurons are noted. Lateral hypothalamic fibers in continuity from the ventral pallidum and amygdala. Scale bar = 2 mm,
diaphorase reactive neurons develop neurofibrillary tangles. Neurofibrillary change has also been described in the hypothalamus in other diseases such as G u a m a n i a n Parkinson's dementia and postencephalitic Parkinsonism. s Hypothalamic lesions have also been described in Huntington's disease brain. Neuronal shrinkage
NADPH diaphorase in human hypothalamus
//
123
and hyperchromasia were found in the tuberoand mamlateral, supraoptic, paraventricular millary nuclei. 35 In the striatum and possibly the cerebral cortex NADPH diaphorase neurons are resistant to damage in Huntington’s disease.’ It is not known if NADPH diaphorase neurons are spared in the human hypothalamus but it is clear that this unique subset of hypothalamic neurons should be studied in neurodegenerative disorders.
/
Acknowledgements-We
Fig. 15. Chart at the level of the mammillothalamic tract (MT) shows prominent cluster lateral to the tract and in posterior hypothalamus. Scale bar = 2 mm.
thank Michelle Forrestall, Steve Conley and Larry Cherkas for photographic skills and Bernard J. Quigley Jr for technical assistance. Thanks to Dr E. P. Richardson for helpful discussions and review of the material. T. Sangruchi is a visiting fellow supported by the Siriraj Foundation, Siriraj Hospital, Mahidol University, Bangkok, Thailand. Supported by NIH grants AGO5134 andNS25588.
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