Journal of Histochemistry and Cytochemistry, Vol. 49, 1453-1468, November 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Atrial Natriuretic Peptide-like Immunoreactivity in Neurons and Astrocytes of Human Cerebellum and Inferior Olivary Complex

James C. McKenziea, Yu-Wen Juana, Charles R. Thomasb, Nancy E.J. Bermanb, and Robert M. Kleinb
a Department of Anatomy, College of Medicine, Howard University, Washington, DC
b Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas

Correspondence to: James C. McKenzie, Dept. of Anatomy, College of Medicine, Howard University, 520 W St, NW, Washington, DC 20059. Fax: 202-265-7055.


  Summary
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Materials and Methods
Results
Discussion
Literature Cited

Atrial natriuretic peptide (ANP) has previously been localized in areas of mammalian brain associated with olfaction, cardiovascular function, and fluid/electrolyte homeostasis. Despite the presence of several types of natriuretic peptide receptors in mammalian cerebellum, neither intrinsic nor extrinsic sources of the natriuretic peptides have been described. In this report we describe the immunohistochemical localization of both intrinsic and extrinsic sources for ANP in human cerebellum. ANP-like immunoreactivity (ANP-LIR) was observed in climbing fibers in the cerebellar molecular layer that probably originated from isolated immunopositive neurons of the inferior olivary complex. Intrinsic sources of ANP-LIR included small subpopulations of protoplasmic and fibrous astrocytes and Bergmann glia, as well as Golgi and Lugaro neurons of the granule cell layer. These results suggest that, in addition to its presumptive roles in local vasoregulation, ANP may serve as a modulator of the activity of Purkinje neurons. (J Histochem Cytochem 49:1453–1467, 2001)

Key Words: ANP, cerebellum, Golgi neurons, astrocytes, Bergmann glia, inferior olive, climbing fibers


  Introduction
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Introduction
Materials and Methods
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Discussion
Literature Cited

NATRIURETIC PEPTIDES and their mRNAs have been localized to forebrain and brainstem regions associated with olfaction, blood pressure regulation, and fluid/electrolyte balance in several species (Jacobowitz et al. 1985 ; Kawata et al. 1985 ; Standaert et al. 1986 ; Netchitailo et al. 1987 ; Ryan and Gundlach 1995 ). Immunohistochemical studies of atrial natriuretic peptide (ANP) distribution have either reported only a few immunoreactive fibers in frog cerebellum (Netchitailo et al. 1987 ) or specifically stated that neither immunoreactive perikarya nor fibers were observed in rat cerebellum (Kawata et al. 1985 ). Other studies have not included the cerebellum (Skofitsch et al. 1985 ; Standaert et al. 1986 ). The distribution of the related peptide, brain natriuretic peptide (BNP), in rat brain has been investigated by Saper and colleagues 1989 , who reported BNP-immunoreactive fibers in cerebellum running parallel to the pial surface and apparently innervating individual Purkinje cells. In addition, only relatively low levels of ANP, as determined by radioimmunoassay, have been detected in the rat cerebellum. The concentration of ANP in cerebellum has been reported to be at or below the level of detectability in rat (Morii et al. 1985 ; Zamir et al. 1986 ), dog (Itoh et al. 1989 ), and pig (Ueda et al. 1988 ; Itoh et al. 1989 ). Standaert and colleagues 1988 have reported relative ANP gene expression levels in rat cerebellum to be the lowest of all brain regions tested. Therefore, no specific endogenous source of natriuretic peptides, particularly ANP, has been identified in the cerebellum to date.

In stark contrast to the above studies, data from human cerebellum demonstrate significant levels of both ANP and BNP (Takahashi et al. 1992 ). However, no localization of ANP or BNP to specific cell types or regions of the human cerebellum has been reported. We have demonstrated sources of ANP in neurons (McKenzie et al. 1990 ) and astroglia (McKenzie 1992 ) endogenous to canine cerebral cortex and have subsequently demonstrated the presence of ANP in neurons and astroglia of human cerebral cortex (McKenzie et al. 1994 ). We now extend those studies to include human cerebellum.


  Materials and Methods
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Materials and Methods
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Five human subjects (see Table 1), received within 3 hr of death, were perfused via the carotid and vertebral arteries with 10% neutral buffered formalin (NBF) as previously described (McKenzie et al. 1994 ). Specimens of cerebellum were blocked and cryoprotected in 35% sucrose and cut at 50 µm on a sliding freezing microtome. Other blocks were sectioned at 50 µm on a vibratome. These sections were immunostained for ANP as described below, embedded in plastic (JB-4; Polysciences, Piscataway, NJ), sectioned at 1–2 µm on a microtome with glass knives, counterstained lightly with toluidine blue, and mounted on glass slides. The method used (frozen or vibratome/plastic embedment) is indicated in the legend of each figure.


 
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Table 1. Case data for specimens

Immunohistochemical staining was performed by the avidin–biotin–peroxidase complex (ABC) technique (Vectastain Elite; Vector Laboratories, Burlingame, CA) using a previously well-characterized antibody against synthetic rat ANP IV (a gift from Dr. Tadashi Inagami) at dilutions of 1:10,000–30,000. Rat ANP-IV with the amino acid sequence H2N-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-COOH (Misono et al. 1984a , Misono et al. 1984b ) was synthesized as previously described (Sugiyama et al. 1984 ), coupled to thyroglobulin, and injected intradermally in Dutch belted rabbits (Tanaka et al. 1984 ). ANP-IV is contained within the rat ANP1–28 sequence and is identical to the low molecular weight form isolated from rat brain (Glembotski et al. 1985 ; Shiono et al. 1986 ). Antisera against synthetic ANP-IV crossreacted 100% with natural ANP-IV, 100% with rat ANP-II, 42.5% with human ANP ({alpha}hANP4–28), and 28% with atriopeptin I, but did not crossreact with arginine–vasopressin, angiotensin I (AI), or angiotensin II (AII) (McKenzie et al. 1985 ).

Free-floating sections were pre-incubated in PBS, pH 7.4, containing 3% normal goat serum (NGS) and 0.3% Triton X-100 for 30 min at room temperature and then transferred to PBS containing 1.5% NGS, 0.3% Triton, and primary antibody (1:10,000–30,000) for 72 hr at 4C. After washing (three times for 5 min in PBS/0.1% Triton), sections were incubated for 30 min in biotinylated goat anti-rabbit IgG (1:200) in PBS/0.3% Triton. The tissue was then washed as above and incubated for 30 min in PBS containing 1% H2O2, washed, and incubated for 30 min in ABC complex in PBS/0.3% Triton. Finally, the sections were incubated for 5 min in 0.05 M Tris buffer (pH 7.2) containing diaminobenzidine (35 mg%) and H2O2 (0.005%) and washed overnight in tapwater. Frozen sections were mounted on glass slides, dehydrated, cleared, and coverslipped. Substitution of normal rabbit serum for primary antibody served as a control. Sections were viewed and photographed in an Olympus Vanox microscope. The ANP IV antibody was further characterized by blot immunostaining. Serial concentrations (1.0 x 10-4–1.0 x 10-11 g) of peptide standards (rat ANP4–28, human ANP1–28, human ANP1–28 antiparallel dimer, BNP-32, rat BNP-45, human CNP: Peninsula Laboratories, Burlingame, CA) were applied to Immobilon PVDF transfer membrane (Milllipore; Bedford, MA) through a 96-well plate and incubated with ANP IV antibody (1:50,000) for 1 hr. Subsequent processing followed the directions given in the Vectastain elite kit (Vector Laboratories). Previous studies have demonstrated that the ANP antibody does not crossreact appreciably with BNP or CNP in absorption controls (McKenzie et al. 1994 ).


  Results
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Materials and Methods
Results
Discussion
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The results of the present study demonstrate conclusively the localization of ANP-like immunoreactivity (ANP-LIR) in specific subpopulations of both neurons and astrocytes in the human cerebellar cortex. Specimens from all cases yielded similar results. There were no obvious differences in the types and sizes of ANP-positive astrocytes found in various regions of the cerebellar hemispheres, flocullus, nodulus, or vermis. Intensity of staining was directly correlated with the concentration of antibody and staining was completely abolished by substitution of non-immune serum for primary antibody. Blot immunostaining revealed maximal reactivity of ANP IV antibody with rat ANP IV and significant reactivity with hANP1–28. There was no apparent reactivity of the antibody with either BNP or CNP (Fig 1).



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Figure 1. Blotting and staining of natriuretic peptides was performed as described in the text. (A) Rat ANP IV (ANP1–25); (B) human ANP1–28; (C) human ANP antiparallel dimer; (D) porcine BNP32; (E) rat BNP45; (F) porcine CNP. Numbers at left are negative log concentrations (e.g., 5 = 1 x 10-5). Appreciable reaction with ANP IV antibody is seen with only rat ANP IV and human ANP1–28.

Protoplasmic Astrocytes
Immunoreactive astrocytes with many short, thick processes were distributed sparsely throughout the granular layer of the cerebellar cortices (Fig 2A). These astrocytes were similar in appearance to those of the cerebral gray matter (McKenzie et al. 1994 ) but were slightly larger and less compact. Processes of these immunoreactive astrocytes entwined neighboring granule cells and their synaptic glomeruli (Fig 3) and were often observed to approach parenchymal blood vessels (Fig 4). There appeared to be no positional preference within the granular layer because these immunoreactive astrocytes were commonly observed at both the granular layer/white matter and granular layer/Purkinje cell interfaces. The mean size of protoplasmic astrocytes was 175.25 x 101.02 mm, including stained processes. In addition, a layer of immunoreactive puncta was observed surrounding the Purkinje cells and extending slightly into the molecular layer (Fig 5). It could not be unequivocally determined whether these puncta originated from protoplasmic astrocytes, Bergmann glia (see below), or other sources (e.g., cells of Lugaro). Rarely, immunoreactive astrocytes were also observed in the molecular layer in or subjacent to the glia limitans of the cerebellum (not shown). These cells were similar in location and morphology to a subpopulation of immunoreactive astrocytes previously described in human cerebral cortex.



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Figure 2. (A) (Case 5802, frozen section, ansiform lobule.) Typical ANP-LIR protoplasmic astrocyte in the granule cell layer of human cerebellar cortex. M, molecular layer; G, granule cell layer; W, white matter. Bar = 50 µm. (B) (Case 6357, posterior inferior lobule.) A typical ANP-LIR fibrous astrocyte of the cerebellar white matter. Immunoreactive processes could often be observed extending several hundred µm from the cell body. Bar = 125 µm.



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Figure 3. (Case 5886, plastic section.) Processes of an ANP-LIR protoplasmic astrocyte are observed interspersed among small groups of granule cells in this plastic (1-µm) section. Some processes appear to be in close contact with granule cells (arrowheads). Bar = 25 µm.



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Figure 4. (A) A single process from a positive protoplasmic astrocyte of the granule cell layer extends a foot process to a parenchymal microvessel (arrowhead). Bar = 50 µm. (Case 6357, posterior inferior lobule, frozen section.) (B) In this plastic section, several process fragments are observed, one of which closely approaches a parenchymal microvessel (arrowhead). Bar = 50 µm. (Case 5886.) (C) At higher magnification, an immunoreactive process appears to be in close juxtaposition to the abluminal surface of a vascular endothelial cell (arrowhead). Bar = 20 µm.



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Figure 5. (A) In all regions of the cerebellar cortex, punctate or globular immunoreactive structures (small arrowheads) were observed associated with the Purkinje cell zone (P) and the adjacent inner molecular layer (M) (Case 6357, frozen section, paraflocculus). Bar = 40 µm. (B) At least some of the immunoreactivity may be attributable to astrocytic process penetrating from the granule cell layer (G). (Case 6357, frozen section, posterior inferior lobule.) Bar = 40 µm.

Fibrous Astrocytes
Immunoreactive astrocytes identical in morphology to those observed in human cerebrocortical white matter were scattered very sparsely throughout the cerebellar white matter (Fig 2B). These ANP-positive astrocytes were characterized by the length of their fine processes, which occasionally appeared to possess varicosities. The mean size of fibrous astrocytes, including stained processes, was 338.57 x 110.71 mm.

Bergmann Glia
The most numerous subpopulation of ANP-immunoreactive astrocytes in human cerebellum was identified as Bergmann glia, based on the position of their cell bodies near the granular layer/Purkinje cell interface and the extension of feather-like processes across the width of the molecular layer to the pial surface (Fig 6).



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Figure 6. (A,B) (Case 6357, frozen section, posterior inferior lobule.) Immunoreactive Bergmann glia were the most numerous glial type observed and were distributed through all regions of the cerebellar cortex. The cell body (arrowhead) was generally situated at the granule cell/Purkinje cell interface and the radial processes extended through the molecular layer to reach the pial surface. The very spinous processes appeared to be organized in columns (A) or in a fan shape (B), dependent on the section orientation. m, molecular layer. Bars = 100 µm.

Neurons
Sparsely distributed ANP-immunoreactive neurons were observed in all regions of the human cerebellar cortex. Although relatively few of each type were observed, ANP-immunoreactive neurons fell into two categories based on morphology and orientation within the granule cell layer. The most numerous, the typical cerebellar Golgi neurons, were frequently found in the granular layer near the Purkinje cell zone but were also observed more centrally disposed within the granule cell layer or occasionally near the white matter. The soma was generally polygonal, with four or more primary dendritic branches. Although the origin of the axon was not identifiable, many delicate, finely beaded collaterals were observed in the area surrounding the soma, extending for variable distances throughout the granule cell layer and occasionally penetrating adjacent folia (Fig 7). Often these immunoreactive collaterals occupied as much as one half of the cross-sectional area of a single folium. A presumably related neuron type was moderately to intensely immunoreactive, possessed an oval soma of medium size, and was always observed in the granular layer immediately subjacent to the Purkinje cell layer (Fig 8). The long axis of the soma and its major dendritic process was oriented parallel to the interface of the granular and Purkinje cell layers. Secondary dendritic processes and a profusion of varicose axon collaterals extended deeply into the granular layer, while a few axon collaterals branched among the Purkinje cells. These neurons are morphologically similar to those known as the cells of Lugaro (Fox 1959 ). No ANP-LIR was observed in neuronal perikarya or processes associated with the deep cerebellar nuclei.



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Figure 7. (Case 6357, frozen section, posterior inferior lobule.) (A) Small subpopulations of ANP-LIR Golgi neurons were observed throughout all regions of the cerebellar cortex. The multipolar cell body was centrally disposed in the granule cell layer and immunoreactive axon collaterals ramified among the granule cells, often extending several hundred µm from the cell body and occasionally penetrating adjacent folia. Bar = 100 µm. (B) In addition to the varicose axon collaterals, smooth dendrites were frequently observed extending into the molecular layer (arrowhead). Staining was generally more intense in the proximal portion of the dendritic arborization and decreased distally. Bar = 50 µm.



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Figure 8. (Case 5550, frozen section, ansiform lobule.) Immunoreactive staining for ANP was also observed in a small number of cells of Lugaro, a subclass of Golgi neurons. These cells were typically unipolar in appearance, located near the Purkinje cell layer, and the primary dendrite was oriented parallel to the granule cell/Purkinje cell interface. Many immunoreactive axon collaterals ramified throughout the granule cell layer (g). Bar = 50 µm.

Immunoreactive climbing fibers were occasionally observed following the trajectory of Purkinje dendritic arborizations in the molecular layer (Fig 9). Immunoreactive fibers were not routinely identified in the cerebellar white matter, and climbing fibers could not be unequivocally identified in the granule cell layer. However, the presence of immunoreactive fine varicose fibers branching in concert with, and following a similar path to, the Purkinje cell dendritic arborization characterized these processes as climbing fibers. Relatively few (one or two) ANP-LIR climbing fibers were observed in each section. These fibers most likely originated in a population of ANP-IR neurons sparsely distributed in all components of the inferior olivary complex, including the medial and superior accessory nuclei (Fig 10). The numbers of immunoreactive inferior olivary neurons appeared to correspond closely to the number of ANP-positive climbing fibers. Fig 11 shows the distribution of ANP-positive neurons, fibrous and protoplasmic astrocytes, and Bergmann glia in a camera lucida drawing of a typical frozen section.



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Figure 9. (Case 6357, frozen section, posterior inferior lobule.) A limited number of varicose immunoreactive climbing fibers were observed in close juxtaposition to the dendritic arborization of individual Purkinje cells, which stand out slightly from background in this micrograph (nonspecific staining). P, Purkinje cell layer. Bar = 70 µm.



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Figure 10. (Case 6357, frozen section, inferior olivary complex.) (A) An intensely immunoreactive neuron is observed in the superior (dorsal) accessory nucleus of the inferior olivary complex. Both axon collaterals and dendritic branches are observed to ramify locally. Bar = 65 µm. (B) A group of immunopositive neurons is observed in the main nucleus of the inferior olivary complex. Typical rosette terminals (arrowheads) are observed adjacent to several immunonegative neurons. Bar = 65 µm.



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Figure 11. Camera lucida drawing of typical frozen section of cerebellum indicating location of ANP-positive cells. The black boxes are Bergmann glia, the "Xs" are protoplasmic astrocytes, the "#" represents a fibrous astrocyte, the "Gs" are Golgi neurons, and the "cf" represents climbing fibers.


  Discussion
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Materials and Methods
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The demonstration of functional natriuretic peptide receptors behind the blood–brain barrier in the mammalian cerebellum virtually demands sources of natriuretic peptides either within the cerebellum itself (intrinsic) or elsewhere within the central nervous system (extrinsic). The results of the present study demonstrate the cellular localization of ANP-LIR within the human cerebellum and strongly suggest that at least some cerebellar ANP is synthesized intrinsically, while one extrinsic source is located in the inferior olive.

ANP-LIR, as detected in the present study, represents localization of ANP and not one of the other natriuretic peptides (BNP, CNP) because results of blot immunostaining revealed no apparent crossreactivity of ANP IV antibody with either BNP or CNP, even at relatively high peptide concentrations. The antibody was raised against a purified synthetic ANP IV and its specificity and sensitivity have previously been well characterized in various tissues (Tanaka et al. 1984 ; McKenzie et al. 1985 , McKenzie et al. 1990 , McKenzie et al. 1991 ). Furthermore, the results of the present study confirm the previous detection of ANP in human cerebellum by RIA (Takahashi et al. 1992 ) and provide data on specific localization.

In contrast to our current results in human cerebellum, previous studies of ANP distribution in rat brain have failed to demonstrate ANP-positive cells (Kawata et al. 1985 ; Skofitsch et al. 1985 ; Standaert et al. 1986 ), significant levels of peptide (Kawata et al. 1985 ; Zamir et al. 1986 ), or appreciable quantities of ANP mRNA (Standaert et al. 1988 ) in the cerebellum itself, although ANP mRNA has been reported in the deep cerebellar nuclei (Langub et al. 1995 ). However, Fujio and co-workers (1987) have detected low to moderate levels of ANP in canine cerebellum by radioimmunoassay. Of most importance for the present study, significant quantities of ANP have been detected in human cerebellum, as previously noted (Takahashi et al. 1992 ).

In addition to ANP, other members of the natriuretic peptide family have been detected in the cerebella of various species. Low levels of BNP have been reported in porcine cerebellum (Ueda et al. 1988 ) and appreciable quantities of BNP have been reported in human cerebellum (Takahashi et al. 1992 ). The third member of the natriuretic peptide family, C-type natriuretic peptide (CNP), has been detected in significant levels in porcine cerebellum (Ueda et al. 1991 ) and at highest concentrations in the hypothalamus and cerebellum of rat brain and in moderate concentration in human cerebellum (Komatsu et al. 1991 ). Totsune and colleagues 1994 have demonstrated both high and low molecular weight forms of CNP in human cerebellum. Langub and co-workers (1995) have demonstrated hybridization of CNP mRNA in neurons of the Purkinje layer and granule cell layer of rat cerebellum. The observed differences in localization most likely result from species variability in expression of the natriuretic peptides.

The presence of intrinsic or extrinsic sources of ANP-like peptides in mammalian cerebellum was originally suggested by several studies reporting species-specific localization of ANP-binding sites. Binding sites for ANP have been reported to be absent (Mantyh et al. 1987 ) or low (Gibson et al. 1986 ) in rat cerebellum but very dense in the granule cell layer of the guinea pig cerebellum (Mantyh et al. 1987 ) and in the monkey cerebellum (Quirion et al. 1986 ; Quirion 1988 ). Specific ANP receptor subtypes have been identified in the cerebellum. Wilcox and colleagues 1991 reported in situ hybridization of mRNA for both of the guanylate cyclase-linked receptor subtypes, ANPR-A and ANPR-B, in monkey cerebellum. Localization of the mRNA for the third receptor subtype, the C or clearance receptor (ANPR-C), appeared to be restricted to Bergmann glia. Konrad and colleagues 1992 have identified a low concentration of high-affinity ANPR-B receptors (CNP selective; Koller et al. 1991 ) in rat cerebellum, while Hernandez and colleagues 1994 demonstrated functional ANP-A receptors in guinea pig cerebellum. Therefore, there appears to be a species-specific heterogeneity in both natriuretic peptides and specific receptors in mammalian cerebellum, with a positive correlation between the presence of peptide and receptors.

A most interesting result of the present study was the localization of ANP-LIR in climbing fibers of the human cerebellum. This was strongly supported by detection of strong ANP-LIR in the perikarya and axonal processes of neurons in the inferior olive. It has long been established that the inferior olive is the sole source of cerebellar climbing fibers (Desclin 1974 ). The presence of ANP-LIR in only a small subpopulation of inferior olivary neurons and climbing fibers is consistent with the heterogeneity of peptide localization in other neuronal networks such as the retina (Blute et al. 2000 ), in which ANP is localized to a small, sparsely distributed subpopulation of amacrine cells.

Localization of ANP in Golgi and related Lugaro neurons of the granule cell layer, in addition to climbing fibers, suggests that ANP may play a role as a neurotransmitter or neuromodulator in regulation of Purkinje cell function. A similar role has been proposed for ANP in the hypothalamus, where ANP alters the activity of vasopressinergic neurons (Samson 1985 ; Crandall and Gregg 1986 ; Standaert et al. 1987 ). Golgi neurons are GABAergic (Storm-Mathesin et al. 1983 ; Aoki et al. 1985 , Aoki et al. 1986 ) and inhibit Purkinje cell activity indirectly by inhibiting the activity of granule cells. Therefore, ANP is found in a position where it can modulate the inhibitory effects of GABA on granule cells. It also may alter the effects of other neurotransmitters released by mossy fibers and/or other inputs.

ANP is unique in its localization to a subpopulation of inferior olivary complex neurons and climbing fibers that persists through adulthood in human brain. Aspartate, an excitatory amino acid, is presumed to be the primary neurotransmitter in climbing fibers (Nadi et al. 1977 ; Perry et al. 1977 ; Wiklund et al. 1982 ). Calcitonin gene-related peptide (CGRP) is expressed transiently during development in olivary neurons and climbing fibers (Yamano and Tohyama 1994 ). Angiotensin II (AII) has been localized in fibers surrounding Purkinje perikarya in rat cerebellum (Changaris et al. 1978 ), but the presence or absence of AII in the perikarya of olivary neurons was not reported in that study. Interestingly, AII has been shown to depress Purkinje cell firing rate and to potentiate the depressant effects of GABA on Purkinje cell function (Tongroach et al. 1984 ). Another neuropeptide, corticotrophin-releasing factor (CRF), is expressed in the entire population of inferior olivary complex neurons and climbing fibers in the adult human brain (Powers et al. 1987 ), as well as in other species (Young et al. 1986 ; Cha and Foote 1988 ; Cummings et al. 1988 ). Furthermore, CRF enhances the Purkinje cell excitatory response to aspartate and attenuates the depressant response to GABA (Bishop 1990 ). Therefore, ANP may play a similar role to CRF and AII in modulating Purkinje cell function, most likely through the activation of guanylyl cylase-linked receptors and increased synthesis of cGMP (Hernandez et al. 1994 ). A modulatory heterogeneity produced by the presence of CRF in various combination with other neuropeptides such as ANP and AII, in subpopulations of climbing fibers may expand the possibilities for regulation of long-term depression (LTD) in cerebellar memory, as proposed by Ito 1986 .

In the present study, ANP-LIR was also localized in protoplasmic, fibrous, and Bergmann astrocytes of human cerebellum. Previously, we have demonstrated similar subpopulations of ANP-positive astrocytes in canine (McKenzie 1992 ) and human cerebral cortices (McKenzie et al. 1994 ). Various neuropeptides have previously been detected in cerebellar astroglia. Both endothelin, a potent vasoconstrictor and antagonist of ANP, and endothelin mRNA have been demonstrated in primary cultures of juvenile rat cerebellar astrocytes (MacCumber et al. 1990 ). Cerebellar astrocytes have also been shown to express the opiate peptide met-enkephalin transiently during development (Zagon et al. 1985 ; Vilijn et al. 1988 ). The results of the present study add support to the presence of heterogeneity in neuropeptide expression by astrocytes (Schwartz and Taniwaki 1994 ).

At this time, the functional significance of ANP in cerebellar astroglia remains speculative. We have previously suggested that ANP synthesized in astrocytes of the canine and human cerebral cortices (McKenzie 1992 ; McKenzie et al. 1994 ) may be released into the interstitial perivascular space, where it may have a direct effect on cerebrovascular hemodynamics or vascular permeability through interaction with abluminal receptors, or that it may act indirectly through effects on cerebrovascular innervation (Nakao et al. 1992 ). Although the relationships between ANP-positive protoplasmic and fibrous astrocytes and parenchymal blood vessels are not as obvious in cerebellum as they are in cerebral cortex, it seems likely that local vasoregulation may be one function of endogenous cerebellar ANP.

The natriuretic peptides may play a role in the regulation of development and cell growth in the cerebellum. ANP has been shown to inhibit mitogenesis and cell growth in several systems, including mesangial cells (Appel 1990 ), vascular smooth muscle cells (Itoh et al. 1990 ), and endothelial cells (Itoh et al. 1991 ). Significantly, both ANP and BNP have been shown to inhibit proliferation and incorporation of [3H]-thymidine in astrocytes cultured from fetal rat diencephalon (Levin and Frank 1991 ). Because the antimitogenic mechanism apparently involves the C receptor (ANPR-C) (Levin and Frank 1991 ), it is possible that ANP-LIR Bergmann glia, which possess the C receptor (Wilcox et al. 1991 ), may regulate proliferation of neighboring glia or neurons via paracrine/autocrine secretion. Therefore, the natriuretic peptides may serve as antagonists to cell proliferation stimulated during development or reaction to injury by other peptides or growth factors, such as endothelin (MacCumber et al. 1990 ) or enkephalin (Zagon et al. 1985 ).

We have demonstrated both neuronal and glial intrinsic sources of ANP-LIR in human cerebellum as well as an extrinsic source in climbing fibers originating from the inferior olivary complex. The various roles of cerebellar ANP derived from these sources, as well as regulatory mechanisms, remain to be elucidated by electrophysiological and molecular biological studies, but are likely to include both local vasoregulation and modulation of Purkinje cell output.


  Acknowledgments

Supported in part by a Howard University Faculty Research Support Grant (to JCM), AHA Heartland Affiliate (to RMK), and NIH grants HL 45241 (to JCM) and P30NS32399 (to NEJB and RMK).

Received for publication October 25, 2000; accepted May 23, 2001.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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