Peptides, Vol. 1, Suppl. 1, pp. 3-10. Printed in the U.S.A.
Exploration of Peptidergic Pathways in Brain by Immunocytochemistry:
A Ten Year Perspective
E A R L A. Z I M M E R M A N , L A U R E N K R U P P , D O N A L D L. H O F F M A N , E L I Z A B E T H M A T T H E W A N D G A J A N A N N I L A V E R
Laboratory of Neuroendocrinology, Department of Neurology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, N Y 10032
ZIMMERMAN, E. A., L. KRUPP, D. L. HOFFMAN, E. MATTHEW AND G. NILAVER. Exploration of pep- tidergic pathways in brain irnmunocytochernistry: A ten year perspective. PEPTIDES 1: Suppl. 1, 3-10, 1980.-- Immunocytochemical techniques have been employed to map specific peptidergic pathways in brain for the last decade. More than 20 different pathways have been described and many more are anticipated. A new chemical neuroanatomy is evolving. Given adequate controls for specificity and improvements in sensitivity, the inherent insurmountable limitation of the technique that remains is the inability to precisely determine the full chemical nature of the substance visualized. The antigenic determinants seen may be localized in the peptide in question, its precursor molecule, or another peptide which is unrelated and often unknown. Biochemical analysis is necessary to solve this problem, but present techniques do not offer the in situ determination provided by immunocytocbemistry (i.e., which neuron contains the peptide?). Some assurances of the nature of the peptidergic system visualized have come from the application of several different antisera to different sequences of the peptide, its precursor molecule, and other peptides derived from it. The vasopressin/oxytocin/neurophy- sins pathway and the ACTH/[3-LPH/a-MSH/fl-endorphin systems are therefore best known because a variety of antisera are available to study them. Discovery of the precursors of other brain peptides in the future will permit a similar approach and provide further evidence that a particular neuron has the capacity to produce a certain peptide.
IMMUNOCYTOCHEMICAL methods have now been em- ployed to study brain pathways for a decade. This has been an exciting period because the possibility of defining the anatomical substrate of a great number of specific pathways has been realized. Work in the previous decade using the Falk-Hillarp fluorescence technique to demonstrate the organization of the biogenic amine systems was the first simultanous demonstration of structure and biochemical (synthetic) function. During this period immunological probes developed for radioimmunoassay were employed in immunocytochemical methods to study hormones in the anterior pituitary gland. Progress in the biochemical charac- terization of posterior pituitary and hypothalamic peptides provided purified or synthetic peptide antigens which were then used to produce antibodies to them primarily for radioimmunoassay. Application of these antisera in im- munocytochemical procedures on brain tissues in the early 1970's not only helped unravel the organization of neurosec- retory pathways, but provided experience with a methodol- ogy which has proven very useful in mapping a host of pep- tides and protein antigens in neurons as well as glia.
The purpose of this chapter is to share some of our expe- rience and that of our colleagues concerning the use of im- munocytochemical probes in the brain over the last decade including successes and failures and some directions for the
future. A complete up to the minute review of brain peptides now seems impossible in this rapidly moving field in the printed paper medium. Such an update may be facilitated by the use of computer technology. The reader is referred to other chapters in this symposium and several excellent re- views [4, 5, 16]. This chapter will focus on the vasopres- sin/oxytocin/neurophysin system and pathways producing ACTH/fl-lipotropin (fl-LPH) and their products because of the authors' experience with them, and their unique position among the many peptide pathways mapped at present. We feel more certain about their visualization because work on their biosynthesis has provided multiple antigens derived from them for localization in the same neurons. In addition, present knowledge suggests that with regard to these two systems, neuronal cell bodies of origin are localized to one region of the brain, the hypothalamus, compared to other peptidergic systems which appear to have perikarya in many different regions. Projections of these two pathways travel to many different brain regions, many of them the same, to form a "hypothalamic brain".
Application of lmmunocytochemical Methods to Study the Neurophysin, Vasopressin, and Oxytocin System
Our present view of the vasopressin and oxytocin sys-
tems is greatly expanded beyond the neurosecretory compo- nent from hypothalamus to posterior pituitary gland which is where it stood in 1970 prior to immunocytochemical studies. These studies began with application of antisera to the neurophysins, so-called "carrier proteins" of the hormones vasopressin and oxytocin [8,26]. The neurophysins are usu- ally easier to localize so that their localization has generally preceded localization of the corresponding hormones. This has led to new discoveries and ultimately one or the other hormones was generally found in a new neurophysin- containing pathway. The reason for this discrepancy is not yet fully clear though there are several good reasons for it. Proteins in general are more easily studied by im- munocytochemistry than the smaller peptides. They are more antigenic and usually result in the preparation of higher quality antisera, and are possibly more stable in tissues with lesser amounts being lost during processing. There is also the possibility that the hormones are further processed to non- reactive products such as the conversion of oxytocin to melanocyte stimulating hormone release inhibiting factor, MIF. Whatever the reason, the best antiserum to neurophy- sin works very well in the immunoperoxidase technique at 1:10,000, while the one to vasopressin must be used at 1:1,000 which usually ends up being the ideal dilution for most whole antisera to any peptide (e.g. oxytocin, LH-RH, substance P) in the peroxidase-antiperoxidase (PAP) tech- nique, whether incubated for 2 or 24 hours. In the early years much higher concentrations of antisera, such as 1:50, were used with peroxidase conjugates of second antibody or un- labeled peroxidase bridge technique, dilutions which had been traditionally used with immunofluorescence. With the development of the more reliable and sensitive PAP tech- nique [18], much higher dilutions were realized which virtu- ally eliminated background staining in most cases and set the stage for more sophisticated absorption experiments with smaller amounts of antigens. Immunological principles, learned previously by radioimmunoassayists, began to be employed in immunocytochemistry: dilution past other usu- ally unknown antibody noise in the system (background), longer incubation times, and competition experiments with homologous antigens and possible cross-reacting fragments and analogs. By the mid 1970's controls using preimmune serum alone were no longer considered adequate. Absorp- tion, at least with synthetic or highly purified homologous antigens was more convincing. Another important set of con- trols were absorptions with analogs and fragments of the homologous antigen in order to determine which amino acid Sequences in the substances were being seen. Unfortunately this is not yet done very often in immunocytochemistry, in part due to the general unavailability of peptide fragments and the tedious nature of these controls. There had also been a tendency to extrapolate from radioimmunoassay data on antigenic determinants to immunocytochemistry for a par- ticular antiserum. In many cases this is probably a dangerous assumption to make. Not only are the dilutions generally used very different, so that a different population of antibodies are being investigated, but the radioimmunoas- sayist controls what is seen by the radiolabel, while the im- munocytochemist has to contend with every possible cross- reacting antigen in the tissue. Furthermore as previously pointed out by Sternberger and colleagues, im- munocytochemistry can be more sensitive than radioim- munoassay. For example, we discovered an antibody to oxytocin by immunocytochemistry using differential ab- sorption experiments with oxytocin and vasopressin against
an antiserum to vasopressin. This was not revealed by radioimmunoassay using the same dilution of antiserum and radiolabels of both hormone antigens [17].
False Positive Results
It seems appropriate at this point to recount some false positive mistakes we have made, some caught before publi- cation and others afterward to illustrate the need for adequate controls. One is the need to control the findings in all parts of a particular system, particularly if a new group of cells is found. We reported neurophysin and oxytocin in human infundibular neurons [5] which turned out to be false. Although we lacked neurophysin for absorption, had we ab- sorbed with oxytocin or used preimmune serum as we had done elsewhere in the brain it would have been evident that the punctate staining was false. The tip-off came from similar observations in the nucleus basalis and substantia nigra of human brain and other areas rich in lipofuscin (R. Defendini, personal communication) which apparently has its own in- trinsic peroxidase activity and reacts with the substrate alone (in this case 3, 3' diaminobenzidine). Lipofuscin is more prevalent in certain neurons in aging brain as used in this case, in the human material and is usually not seen in younger brains which one tends to use in experimental animals. Another unpublished observation of the author was the localization of somatostatin in large motor neurons of the brainstem of the rat. Large motor neurons are always sus- pect because they frequently react in a non-specific way with a variety of antisera. Preimmune serum was also found to react. Preabsorption with somatostatin eliminated reactivity in the hypothalamus in known areas such as the zona externa of the median eminence, but had no effect on the large motor neurons which we concluded was a false localization. An- other early mistake caught before publication was the appar- ent localization of luteinizing hormone-releasing hormone (LH-RH) in the magnocellular system (G. P. Kozlowski and E. A. Zimmerman, unpublished observation). Absorption with LH-RH only eliminated most zona externa reactivity and that found in a few infundibular neurons. Reactivity in the supraoptic and paraventricular pathways to the posterior pituitary zona externa remained. The original immunogen was bovine serum albumin (BSA) conjugated to synthetic LH-RH. The possiblity therefore existed that in this antiserum there were antibodies to BSA which reacted in the magnocellular system. This was confirmed by abosrption with BSA. Apparently these neurons contain albumin or something that looks like it. We have not had similar prob- lems with conjugates of or antibodies to hemocyanin or bovine thyroglobulin except for the discovery by radioim- munoassay (A. Liotta, D. T. Krieger, E. A. Zimmerman, unpublished) of a small amount of antibody to ACTH in an antiserum (not cross reactivity with LH-RH which shares a dipeptide sequence with ACTH) raised to LH-RH conju- gated to bovine thyroglobulin. Radioimmunoassay of the original bovine thyroglobulin used for conjugation to LH-RH revealed a small but significant amount of contamination with ACTH. In the near future the production of monoclonal antibodies in tissue culture by hybridomas is expected to eliminate a lot of these problems. For the time being how- ever, the above concerns and some to follow are important for immunocytochemists and those interpreting their data.
Two of the authors' now controversial reports warrant further discussion here. One was a report of neurophysin in tanycytes initially reported in monkeys [14] which we could
BRAIN PEPTIDERGIC PATHWAYS 5
not reproduce in this or other mammalian species until we dehydrated rats for other reasons and found it [27]. In retro- spect the earlier monkeys reported had diarrhea and were culled from an imported colony. They were probably dehy- drated. The physiological state of the experimental animal is important in this case.
The other issue was our earlier report of the apparent presence of vasopressin in some oxytocin cells [17,27]. Con- trois using differential preabsorption of antisera to oxytocin and vasopressin against both synthetic hormones suggested specificity [17]. We knew that the antiserum to vasopressin contained a small amount of cross-reacting antibody to oxytocin, and a small amount of specific anti-oxytocin which was determined in homozygous Brattleboro rats with diabe- tes insipidus [17]. In normal rats, oxytocin preabsorption had no apparent effect on the antiserum to vasopressin. Other investigators, however, using antisera to these hor- mones differentially absorbed by solid phase methods re- ported no evidence for both hormones in the same cell [20,22]. In the last year we have succeeded in raising specific antisera to oxytocin and vasopressin, and to date we have also found no evidence for the presence of both hormones in the same cell (G. Nilaver, D. L. Hoffman and E. A. Zim- merman, Fig. 1). In this situation application of solid- phase-affinity-purified antibody is a better method of resolu- tion than preabsorption.
Angiotensin H and the Magnocellular System
Phillips and colleagues [13] and our own laboratory have recently reported immunocytochemical evidence for angiotensin II (All) in the supraoptic and paraventricular nuclei of the hypothalamus [6]. Although our recent results are still in the preliminary stage, they are instructive from technical and scientific points of view, and suggest that what is being visualized may not be real peripheral rat AII. Preab- sorption experiments with 5-isoleucine All (normal per pheral rat AII) were less complete than those using 5-valine AII. By radioimmunoassay, the antiserum contained two antibodies: one with total cross-reactivity between 5-valine All and 5-isoleucine AII, and another specific for 5-valine All (G. Valiquette, E. A. Zimmerman, unpublished). Affin- ity purification of the antibody with 5-valine AII may resolve the issue. The original immunogen was a 5-valine All conju- gate and it is possible that a specific antibody to 5-valine All in this antiserum prefers the substance visualized in mag- nocellular neurons which is more like 5-valine All than real rat peripheral AII (5-isoleucine).
Several other types of experimental data suggest that the magnocellular AII reactivity is not related to the classical renin-angiotensin system. Instead we appear to be identify- ing something in the vasopressin synthetic pathway, possibly a part of the precursor pro-pressophysin itself [I]. The reac- tivity is found in vasopressin, not oxytocin cells (Fig. 1), including the suprachiasmatic nucleus which is known to produce vasopressin. There is a marked increase in im- munoreactivity in zona externa fibers after bilateral ad- renalectomy known to be associated with increases in vaso- pressin [ 19]. Our more recent studies indicate that it is absent from homozygous Brattleboro rats with diabetes insipidus (DI rats) which cannot produce pro-pressophysin [1]. Biochemical studies are underway to determine if this im- munoreactivity is part of pro-pressophysin. Whether the rat brain actually produces a type of biologically active 5-valine AII, or whether this is simply cross-reactivity remains to be
determined. It does suggest that "the magnocellular AI I" reported may be different from peripheral All in rat. The known shared amino acid sequence of the different opioid peptides: enkephalin, /3-endorphin, and dynorphin, raises the possibility of many shared sequences in different sub- stance in brain and supports the idea that there may be sev- eral angiotensin II molecules in rat. This also brings us to the most serious inherent limitation of the immunological methods. Even with the best controls and the most careful work, immunocytochemistry is not the ultimate chemistry. This is not meant to demean this approach which will con- tinue to be a valuable guide, but its limitations are now being realized and should be taken into account. Im- munocytochemistry is the only means at present to visualize and trace pathways which synthesize and transport peptides. The problem is that one does not know the real chemical structure that is being visualized. One can and should find out which amino acid sequences are being seen by the antibody system, but whether it is actually the substance in question or something else that contains the sequence or sequences which are very similar and react cannot be fully discerned. If one didn't know about oxytocin, many antisera to vasopressin which also see oxytocin would have led to the conclusion that all cells in the supraoptic nucleus for exam- ple, contain vasopressin. Present biochemical methods on the other hand are not designed to demonstrate peptides within specific neurons and their pathways. Obviously the present state of the art requires both approaches.
Neurons of Origin or Uptake?
The above issue as to what is being visualized and a sec- ond one raised recently concerning the source of brain ACTH (and/3-LPH//3-endorphin) have been in great part re- solved by the localization of several parts of the same biosynthetic system within the same neurons and their proc- esses. The association of vasopressin and oxytocin with their respective neurophysins, and ACTH,/3-LPH and their prod- ucts are the best examples of how the immunocytochemical and biochemical approaches have been most complimentary. The finding of specific neurophysins in the neurons produc- ing oxytocin and vasopressin [3,23] is further convincing data that these neurons in the hypothalamus contain the biosynthetic machinery to produce the active hormones which ultimately arrive in the posterior pituitary gland [1] confirming the results of a battle won many years earlier by Ernst Scharrer and colleagues concerning the concept of neurosecretion [15]. A similar controversy was waged in the last decade in reference to ACTH in the brain. It is made in the brain or in the pituitary and transported to brain [10]? The matter now seems resolved in the sense that it is produced in both places, and the presence of im- munocytochemistry on the scene helped in reaching this conclusion. It is still not known, however, how much pitui- tary ACTH or its related peptides actually get into brain.
In the anterior pituitary, ACTH and /3-1ipotropin were found in the same cell which was expected since they are derived from the same common precursor [9]. By im- munocytochemistry they are also found in the same neuronal cell bodies in the arcuate nucleus and around and lateral to the ventromedial nucleus of the hypothalamus. Further- more, antisera to other fragments of the system localize to these cells (fl-endorphin, c~-MSH, and the 16-K part of the precursor; see chapter by Watson for review). An elaborate network of fibers arise from these cells and appear to inner- vate many intra- and extra-hypothalamic structures includ-
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BRAIN PEPTIDERGIC PATHWAYS 7
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B FIG. 1 A, B. Immunocytochemical localization of angiotensin II (AII), vasopressin (VP), and oxytocin (OT) in serial coronal 6/zm sections of a normal rat supraoptic nucleus. In A, (magnification× 125) note that the distribution of All and VP is the same, while OT-containing cells tend to be more dorsal. In B, at higher power (x 275) All and VP are found in the same neurons which lack the OT immunoreactivity (arrow) that is found in different cells (unpublished).
8 ZIMMERMAN ET AL.
FIG. 2. Fluorescence photomicrograph of a fluorescein-labeled protein conjugate of the ben- zodiazepine analog Ro-3072 bound to living mouse melanoma cells in culture. Note the localization of the drug on cell bodies and processes. Preincubation with unlabeled drug prevented uptake of the label. × 1050 (E. Matthew, unpublished).
ing the diencephalon and midbrain. Destruction of the ar- cuate region by administration of monosodium glutamate in rats destroys the system, and hypophysectomy has no effect [7]. It makes no neurobiological sense that these cells would take up all these parts of the precursor at the same time by retrograde portal flow and transport along their elaborate pathways to other brain sites. Furthermore there is now evi- dence that the brain produces the precursor pro-opiocortin
or something very similar to it. In regards to our own im- munocytochemical studies of this system, we suspect that we have been visualizing the precursor. It was determined that we were visualizing C-terminal ACTH and N-terminal/3-LPH [11]. Most brain ACTH appears to be converted to a-MSH (from N-terminal ACTH) and fl-endorphin (from C-terminal fl-LPH). Antibodies which localize a-MSH and not ACTH have been used to localize the arcuate system as well. It is of
BRAIN PEPTIDERGIC PATHWAYS 9
interest that another group of dorsolateral hypothalamic neurons unreactive to ACTH and/3-LPH were found to react to a-MSH which may represent yet another unrelated sys- tem (see chapter by Watson). Again the tendency for repeti- tion of the same or similar antigenic determinants in different peptides makes interpretation of immunocytochemical re- sults most tentative until other supportive data can be ob- tained, and immunocytochemistry is clearly dependent on biochemistry. Since other parts of the possible precursors of all the other more than 20 peptides mapped in brain are un- known, although important, these results must be considered preliminary.
Even the two systems we think we know the best may well undergo modification. Some of the vasopressin, oxytocin, or neurophysin reactive neurons may yet turn out to contain vasotocin or some other unknown products.
False Negat ives
False negatives are as big a problem as false positives. Gradual appreciation of the complexities of the vasopres- sin/oxytocin-neurophysin system is a good example. Work from other laboratories in the late 1970's revealed a remark- able extrahypothalamic projection system which appears to arise from the paraventricular nucleus [12,20]. This was to- tally missed by those of us focusing on hypothalamus and diencephalon. As is probably true in most of science, one gets caught up in one little area and tends to see what one wants to. It is going to take many more years to sort out this system which takes one all over the brain. We have seen occasional neurophysin and oxytocin-positive fibers even in cerebral cortex. It may even turn out that there are cell bodies elsewhere, such as a few we have seen reactive to neurophysin in temporal lobe, but not enough to adequately test or be sure of. Application of colchicine to increase perikaryal localization has been useful in some systems, but it has not revealed other cell bodies to date in the magnocel- lular system. Nevertheless it would not be surprising if further studies with better probes reveal even more details for this system than previously realized.
Future Directions
In addition to these limits and hopes for peptide im-
munocytochemistry in the future including the im- provements in specificity and larger quantities of antibodies promised by the monoclonal techniques, the possibility of combining immunocytochemistry with receptor localization now seems an exciting new direction. Localization of pep- tides on probable receptors on target neurons was recently shown in brain for neurotensin [24]. Combinations of these techniques with immunocytochemistry would allow simulta- neous visualization of fibers transporting peptides and func- tional target sites. The technical aspects of this, however, will probably be difficult. Immunocytochemistry at the ul- trastructural level is only beginning to reveal the nature of peptide synapses [2]. Retaining ultrastructural detail while maintaining the receptor probe will not be easily solved. New receptor probes other than radioisotopes may be help- ful. We recently developed one for the benzodiazepines which holds promise for ultrastructural analysis. Antibody to hemocyanin was conjugated to a benzodiazepine analog which attaches to receptors on living melanoma cells as de- termined by fluorescence microscopy (Fig. 2). How this will behave at the ultrastructural level with the addition of hemocyanin, and whether it can be used for tissue sections remains to be determined. In addition to combinations with chemical and physiological methods, further directions in immunocytochemistry will also include its use with other anatomical methods in order to define the interconnections of specific pathways now being determined with other spe- cific systems. Methods need to be perfected both for simul- taneous tracing of pathways and for specific innervation. For example the paraventricular nucleus both projects to and receives projections from a variety of brain sites including regions in the brainstem thought to contain biogenic amines. We have reached the point where such transmitter-specific anatomical interconnections need to be worked out in order to more completely understand the role of peptidergic path- ways in the brain.
ACKNOWLEDGEMENTS
The authors thank Dr. Susan Rosario for technical assistance, Mrs. Aida Laude for help with the manuscript, and Hoffmann- LaRoche for Ro-3072. Supported by NIH Grants AM 20337, HD 13147, and HL 24105.
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