Ultrastructural relations between β-adrenergic receptors and catecholaminergic neurons

Brain Research Bulletin, Vol. 29, pp. 257-263, 1992

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Ultrastructural Relations Between ,&Adrenergic Receptors and Catecholaminergic Neurons CHIYE AOKI*’ AND VIRGINIA

M. PICKEL’r

*Center for Neural Science and Biology, New York University, New York, NY 10003, and fDivision of Neurobiology, Department of Neurology and Neuroscience, Cornell University Medical College, New York, NY 10021

Received 27 November 199 1 AOKI, C. AND V. M. PICKEL. Ultrastructural relations between fl-adrenergic receptors and catecholaminergic neurons. BRAIN RES BULL 29(3/4) 257-263, 1992.-We performed dual electron microscopic immunocytochemistry to determine the precise cellular relations between @-adrenergic receptors (PAR) and catecholaminergic terminals within adult rat brains. An antibody,

PAR404, against a peptide corresponding to the C-terminus of the hamster lung BAR (& subtype) together with an anti-tyrosine hydroxylase(TH), a catecholaminergic marker, were used. Results show predominant labeling for BAR404 within small astrocytic processes (P-A). This is in sharp contrast to earlier results which showed neuronal labeling when using antibodies against the third intracellular loop of the receptor and of neurons-plus-astrocytes labeled using antibodies against the whole @AR molecule. /3-A within visual cortex and nuclei of the solitary tracts frequently contacted blood vessel basement membrane and TH-immunoreactive terminals. TH-immunoreactive axons forming axo-axonic juxtapositions with non-TH terminals were also noted to be surrounded by P-A. In the area postrema, a brain region lacking a blood-brain barrier, few /3-A occurred adjacent to TH-immunoreactive terminals or elsewhere. Thus, I) catecholamines may act beyond morphologically identifiable synapses; 2) /3-A may mediate interactions between catecholamines and other transmitters; 3) there may be substantial heterogeneity in the structure or the conformation of the PAR protein between neurons and glia or across CNS regions. P-Adrenergic receptors &Adrenoceptors Nucleus of the solitary tract Astrocytes Visual cortex Catecholamines Ultrastructure Blood-brain barrier Synapse Area postrema

AT least two factors dictate the site of action of neurotransmitters: the site of release of the neurotransmitter and the location of appropriate receptors. With regard to catecholamines, elegant electrophysiological studies have shown that volume transmission, rather than discrete synaptic transmission, occurs where sympathetic nerve fibers innervate smooth muscles of blood vessels (3 1). Within the CNS, previous immunocytochemical results (26) indicate that more than half of the noradrenergic axons form identifiable synaptic junctions. On the other hand, Descarries and his colleagues, who have localized monoamine uptake sites and synthesizing enzymes, propose that catecholamines in the cerebral cortex may also be released from portions along axons that are devoid of morphologically identifiable synaptic specializations (8,lS). Our approach has been to use antibodies directed against /3-adrenergic receptors (@AR) to delineate some of the sites of action of norepinephrine. A previous study showed neuronal as well as glial localization of immunoreactivity using antibodies directed against the frog erythrocyte PAR (& subtype) (5.33). Another previous study that used a monoclonal

Area postrema Circumventricular organ

antibody against the third intracellular loop of hamster lung PAR (& subtype) (6,7) showed predominantly neuronal labeling, particularly within cell bodies and proximal dendrites, and little astrocytic labeling. The present study used antibodies directed against amino acids 404 through 4 18 (PAR404) corresponding to the C-terminus of hamster lung PAR (& subtype) having the following sequence: Cys-Leu-Asp-Ser-GlnGly-Arg-Asn-Nle-Ser-Thr-Asn-Asp-Ser-Pro-Leu where Nle is norleucine. This region of the receptor is homologous to the equivalent region of human brain &-adrenergic receptors ( 14). On the other hand, the amino acid sequence of this region is not at all homologous to any region of the &adrenergic receptors ( 17) or of the avian turkey erythrocyte /3-adrenergic receptors (fir subtype) (35). Contrary to our expectation, results show that PAR404 immunoreactivity is localized almost entirely within astrocytes and that the proximity of PAR404 immunoreactive astrocytes to catecholaminergic neurons differs across brain regions. This paper will review the ultrastructural relationship between PAR404 immunoreactive astrocytes and catecholaminergic neurons in three brain regions: the visual cortex, which is a terminal field for noradrenergic

’ Requests for reprints should be addressed to C. Aoki, Center for Neural Science, 6 Washington Place, New York, NY lOOQ3

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FIG. I. Electron micrographs showing the ultrastructural relations between catecholaminergic processes and $AR404 immunoreactive astrocytes near a blood vessel in the NTS of an adult rat. Asterisks in panel A indicate dendrite (left) and terminal (right) within a low magnification electron micrograph which are enlarged in panels B and C (arrows point to the panels that are related to each asterisk). The presence of electron-dense. silver-intensified gold particles within the asterisklabeled profiles indicates that these contain immunoreactivity for tyrosine hydroxylase and are. thus. catecholaminergic. At higher magnification, immunoperoxidase reaction product reflecting PAR404 immunoreactivity is detectable within astrocytic processes. Contacts between PAR404 immunoreactive astrocytes (P-A) and the catecholaminergic dendrite (CD) or terminal (CT) are evident (arrowheads). OAR404 immunoreactivity occurs as noncontinuous patches along the astrocytic plasma membrane, including portions that are not in contact with catecholaminergic processes. For example, the arrowhead pair in B points to gap junctions formed between two astrocytic plasma membrane with little, if any, PAR404 immunoreactivity while the arrowhead pair in C points to the gap junction between two astrocytes with PAR404 immunoreactivity. A low level of PAR404 immunoreactivity is also evident within a vacuole in the pericyte (P = pericyte; E = endothelial cell; bvl = blood vessel lumen in A, and two small arrows in B point to PAR404 immunoreactivity within P), In C. the open arrow points to the postsynaptic membrane associated with an unlabeled terminal (UT) while the closed arrow points to the postsynaptic density associated with CT. Bars = I pm in A, 0.5 pm in B and C.

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OF @AR

FIG. 2. Electron micrographs from serially collected ultrathin sections showing the ultrastructural relations between a catecholaminergic terminal and PAR404 immunoreactive astrocytes in the visual cortex. Electron micrograph in A shows three silver-intensified gold particles over a terminal. Recurrence of these particles in the other ultrathin sections confirm that the process is catecholaminergic (CT). CT forms a type II, symmetric synapse (19) onto an unlabeled dendrite (uD) (arrows in A and B point to the postsynaptic membrane within uD) and is also juxtaposed to a perikaryon (P). A fine astrocytic process exhibiting PAR404 immunoreactivity (asterisks in cytoplasm) courses between CT another synaptic junction formed between unlabeled processes (UT in B and C = unlabeled terminal). To the right of CT are a few unlabeled axons (uA, arrows in B point to two of them) which, unlike UT. are in direct contact with CT. A BAR404 immunoreactive astrocytic process courses next to uA, Bar = 0.5 grn.

(NTS) which contain catecholaminergic cell bodies and terminals; and area postrema (AP), a region lacking the blood-brain barrier and also containing catecholaminergic neurons. Detailed descriptions can be found in the papers by Aoki (1) and Aoki and Pickel (2). neurons;

nuclei

of the solitary

tracts

METHOD

Dual immunocytochemistry performed during this study combined two electron-dense labels that are easily differentiated by electron microscopy: peroxidase reaction product to identify PAR404 immunoreactivity and silver-intensified immunogold particles to identify the enzyme, tyrosine hydroxylase, within

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FIG. 3. Electron micrographs showing ultrastructural relations between a catecholaminergic terminal and astrocytes ot AP. Electron micrographs show immunogold-labeled catecholaminergic terminals (CT) forming synaptic junctions with small (left) and larger (right) dendrites (arrows point to postsynaptic densities). These synaptic junctions are encapsulated by astrocytic processes lacking BAR404 immunoreactivity (asterisks). UT = unlabeled terminal. Bar = 0.5 pm

catecholaminergic cell bodies and processes. Cells and processes immunoreactive for the two markers were detected within aldehyde-fixed Vibratome sections of adult rat (Sprague-Dawley) brains. Details of the method have been described elsewhere (I 2). Anti-@AR404 has been used previously to identify PAR by Western blotting (34). Monoclonal anti-TH was purchased from Boehringer Mannheim.

RESULTS

The boundary between NT’S and AP is one region in brainstem showing the most intense immunoreactivity for PAR404. Another region exhibiting strong immunoreactivity for PAR404 is the supragranular layer of visual cortex. Both of these regions contain high densities of astrocytic processes, as revealed by the immunocytochemical labeling for two astrocytic markers-glial fibrillary acidic protein (GFAP) (9) and glutamate dehydrogenase (3.4). Light microscopic immunocytochemistry revealed that OAR404 immunoreactivity in these three regions was associated with punctate processes that were distributed throughout the neuropil. Perikarya, small and large. were revealed as unlabeled profiles embedded within strongly PAR404 immunoreactive tissue. Following dual labeling for PAR and catecholamines. electron microscopic examination of the three regions revealed that most of the PAR404 immunoreactivity was associated with patches of astrocytic plasma membrane. In agreement with known distributions of catecholaminergic pathways, tyrosine hydroxylase immunoreactivity occurred within neuronal perikarya and dendrites of brainstem. and within axons of brainstem and visual cortex. The fine processes (less than 0.3 hrn wide) within the NTS (Fig. 1) and visual cortex (Fig. 2) that showed BAR404 immunoreactivity were easily identified to be astrocytic based on their irregular contours, paucity of intracellular organelles and gap junctions that formed between them (27). Although PAR404 immunoreactivity was prevalent along distal portions of astro-

cytic processes. large surfaces of plasma membranes near astrocytic somata remained unlabeled. PAR404 immunoreactive astrocytes in the NTS surrounded most of the catecholaminergic cell bodies, dendrites and axons in the NTS (Fig. I). Similarly. the majority of catecholaminergic axons in the visual cortex were juxtaposed to BAR404 immunoreactive processes of astrocytes (Fig. 2). In both regions, noncatecholaminergic neurons in the vicinity of catecholaminergic processes also were contacted by $AR404 immunoreactive astrocytes (Figs. IC and 2). Together. these light and electron microscopic observations indicate that BAR404 immunoreactive distal processes of astrocyte in the NTS and visual cortex are numerous, distributed widely throughout the neuropil and interleaf between catecholaminergic and noncatecholaminergic neurons. Examination of the electron microscopic preparations from the AP showed another type of relationship between catecholaminergic neurons and PAR404 immunoreactive processes. The AP is a highly vascularized brain region. Associated with these blood vessels are numerous astrocytic processes which, unlike those in the NTS. contain a complex array of intracellular organelles. These include structures that resemble the Golgi apparatus. rough endoplasmic reticulum, vacuoles of various sizes and mitochondria. In sharp contrast to the NTS, these astrocytic processes rarely exhibited PAR404 immunoreactivity, even in the vicinity of catecholaminergic dendrites, axons, or terminals (Fig. 3). Conversely, even within planes of section where both @-A and catecholaminergic axons could be identified, the two types of processes rarely occurred juxtaposed to each other (not shown). DISCUSSION

Why does the antibody against BAR404 not recognize neurons? Conversely, why does the antibody against the third intracellular loop of PAR not recognize more astrocytes? One possible interpretation of these results is that the structure or conformation of PAR is different between neurons and glia. The presence of distinct genes for neurons versus glia has already

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FIG. 4. Schematic diagram depicting different types of associations between PAR404 immunoreactive astrocytes. a catecholaminergic axon and dendrites. Blood vessels in AP are formed by fenestrated endothelial cells (e). Processes of astrocytes (a) encircle endothelial cells loosely, thus leaving large volumes of perivascular space (p). These astrocytic processes usually lack PAR404 immunoreactivity and contain vacuoles or vesicles of various sizes. Catecholaminergic terminals that occur in AP form asymmetric synaptic junctions and are not closely associated with PAR404 immunoreactive (fi-ir) astrocytes. In contrast, more of the astrocytic processes in the NTS and visual cortex (to the right of the dotted line) exhibit &ir. These include astrocytic end feet that surround blood vessels as well as those in the neuropil surrounding CA. Astrocytic perikarya usually are without detectable levels of p-ir. Besides CA, /3-ir astrocytic processes occur juxtaposed to other unlabeled axons (uA) and unlabeled dendrites (uD) in such ways as to permit or not permit direct contacts with CA

been demonstrated for another transmitter receptor, i.e., the nonNMDA glutamate receptor within CNS [rev. in (30)]. Differences may, instead, reflect differential posttranslational modifications (e.g., phosphorylation, truncation) (16) or differential binding of /JAR receptor molecule with other intracellular molecules, such as fl-arrestin (24), may have caused stearic hindrance for their recognition by the antibodies. On the other hand, the differences could not have arisen due to alternative splicing, since the PAR gene has no introns (16). Figure 4 summarizes the regional differences in the relations between catecholaminergic axons, PAR404 immunoreactive astrocytic processes and other unlabeled neuronal processes. Juxtaposition of catecholaminergic axons to PAR404 immunoreactive neurons was notable in the NTS as well as in the visual cortex, but rare in AP. Two interpretations are consistent with these observations. First, the position of these astrocytes exhibiting putative sites for catecholamine binding may define the sphere of diffusion for neuronally released catecholamines. If so, then @AR404-immunoreactive astrocytes in the NTS and visual cortex that intervene between catecholaminergic axons and unlabeled axons may prevent axo-axonic interactions, while astrocytes that encapsulate the two types of axons may enhance their interactions. Similarly, PAR404 immunoreactive astrocytes that encircle dendrites together with catecholaminergic axons may facilitate axo-dendritic interactions while other astrocytic processes that intervene between them could prevent such axodendritic interactions. In this way, PAR404 immunoreactive astrocytes may play an active role of determining which neuronal processes within a small neuropilar volume can interact.

Another possible role for PAR404-immunoreactive astrocytes may be to mediate interactions between catecholaminergic and noncatecholaminergic neurons via biochemical pathways that are specific for astrocytes. For example, alterations in astrocytic uptake of L-glutamate and GABA following activation of astrocytic PAR have been demonstrated (see E. Hansson’s paper in this issue). This example shows that catecholamine-receptive astrocytes can mediate interactions between catecholaminergic and noncatecholaminergic neurons by coupling neuronal release of catecholamines to alterations in the extracellular concentration of another transmitter. The biochemical pathways that couple PAR activation to transmitter uptake systems within astrocytes is not currently known. The paucity of contacts between catecholaminergic neurons and PAR404 immunoreactive astrocytes in AP may suggest the converse: namely, that a) the sphere of diffusion of neuronally released catecholamines may be greater in the AP, since the distances between catecholaminergic axons and PAR are greater; and b) astrocytes play a less significant role of mediating interactions between catecholaminergic and noncatecholaminergic neurons via PAR-activation. In addition, the paucity of BAR404 immunoreactive astrocytes near blood vessels of the AP suggest that circulating catecholamines may be able to diffuse relatively freely through blood vessel fenestrations and diffuse through greater distances in the AP neuropil. However, since PAR404 immunoreactive astrocytes are abundant at the NTS-AP boundary, diffusion of circulating catecholamines beyond AP and into the NTS may be minimal. Together, these studies point to the need for further research

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that examines the functional interrelations between catecholaminergic neurons and astrocytes. For example, results from parallel studies in the visual cortex suggest that catecholaminergic axons containing large clusters of vesicles show proximity to fiAR immunoreactive astrocytes, while catecholaminergic axons containing few vesicles occur at a distance greater than three neuronal processes from PAR404 immunoreactive astrocytes ( I). These differences in astrocyte-axon relationships suggest that catecholamine-receptive astrocytes may exhibit positive chemotropism towards sites for catecholamine release. Future ultrastructural studies that combine PAR activation in vivo with BAR immunocytochemistry would be needed to test this possibility. Further work in molecular biology would also help elucidate

AND

PICKEL

or not there are other genes for BAR yet to be cloned that are expressed differentially between neurons and glia and across different regions in the CNS.

whether

A(‘KNOWLEDGEMEN-I-S

We thank Catherine D. Strader for her generous gifts of antibodies against peptides corresponding to the intracellular third loop and Cterminal tail of BAR and Tong H. Joh for the antiserum against the whole PAR. We also would like to thank Strader for her criticisms of the manuscript. We appreciate the help of Adam Starr for the reproduction of electron micrographs. This work was funded by the following grants: EY08055 to C.A.: MH40342 to V.M.P.

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