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5 Spemann, H. (1938) Embryonic Development and Induction, Yale University Press 6 Kelley, D. and Pfaff, D. (1978) in Biological Determinants of Sexual Behavior Hutchison, J. (ed.), pp.225--254, John Wiley and Sons 7 Goy, R. W. and McEwen, B.S. (1980) Sexual Differentiation of the Brain, The MIT Press 8 Lisk, R. (1962) Am. J.Physiol. 203,493-496 9 Pfaff, D. (1968) Science 161, 1355-1356 I0 Stumpf, W. (1968) Science 162, 1001-1003 11 Naftolin, F., Ryan, K. and Petro, Z. (1971)J. Clin. Endocrinol. Metab. 33, 368-370 12 Raisman, G. and Field, P. (1971)Science 173, 731-733 13 Pfaff, D. W. (1980) Estrogens and Brain Function: Neural Analysis of a HormoneControlled Mammalian Reproductive Behavior, Springer Verlag 14 Nottebohm, F., Stokes, T. and Leonard, C. (1976) J. Comp. Neurol. 165,457-486 15 Nottebohm, F. and Arnold, A. (1976) Science

194, 211-213 16 Gurney, M. and Konishi, M. (1980) Science 208, 1380-1383 17 Gurney, M. (1981) J. Neurosci. 1,658--673 18 Gurney, M. (1982) Brain Res. 231,153-172 19 Adkins, E.(1975)J. Comp. Physiol. Psychol. 89, 61-71 20 Nottebohm, F. (1981) Science214, 1368-1370 21 Konishi, M. and Akutagawa, E. in Selective Neuronal D e a t h (Ciba Foundation Symposium 126), The Ciba Foundation (in press) 22 Gorski, R. A., Gordon, J., Shryne, J. and Southam, A. (1978) Brain Res. 148, 333-346 23 Breedlove, S. M. and Arnold, A. P. (1980) Science 210, 564-566 24 Breedlove, S. M. and Arnold, A. P. (1981) Brain Res. 225, 297-307 25 Truman, J. and Schwartz, L. M. (1984) J. Neurosci. 4, 274--280 26 Hamburger, V. (1975)J. Comp. Neurol. 160, 535-546 27 Pittman, R. and Oppenheim, R. (1979) J.

Comp. Neurol. 187,256-277 28 Nordeen, E., Nordeen, K., Sengelaub, D. and Arnold, A. (1985) Science 229, 671-673 29 Wilson, J., George, F. and Griffin, J. (1981) Science 211, 1278-1284 30 Kelley, D . B . (1986) J.Neurobiol. 17, 231-248 31 Kelley, D. B. (1980) Science 207,553-555 32 Sassoon, D. A. and Kelley, D. B. Am. J. Anat. (in press) 33 Breedlove, S. M. (1986) J. Neurobiol. 17, 157-176 34 Breedlove, S. M. (1986) Soc. Neurosci. Abst. 12, pp. 1220 35 Sassoon, D. A., Segil, N. and Kelley, D. B. (1986) Dev. Biol. 113, 135-145 36 Tobias, M. and Kelley, D. B. (1985) Soc. Neurosci. Abstr. 11,496

Darcy B. Kelley is at the Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

Pathways to pmgr s- the rise of modern neu atomlcal hrdques Edward G.Jones The range of neuroanatomicalmethods has undergone an enormous expansion in the last decade. It is remarkable to think that less than 15 years ago a single technique (that of Nauta) dominated neuroanatomical studies. The first of the newly introduced methods caused an explosion in connection-tracing studies. However, the new emphasis in neuromorphology is on the characterization of individual neurons and on experimentation that demonstrates the functional properties of defined classes of cells. In 1963 when I was contemplating a career in research, a distinguished comparative morphologist told me that there was not much point in going into neuroanatomy since about the only thing one could do was invent new techniques. For the next ten years, during which time the Nauta method in its several forms was essentially the only experimental technique available in neuroanatomy, his remark struck me as being singularly odd. However, perhaps he had more perspicacity than I had credited him with, and was looking beyond the silver impregnation of degenerating axons to the future much richer in neuroanatomical techniques that is now with us.

with the Fink-Heimer modification which was itself further modified1. To many, the replacement of these methods of tracing axonal connections by one based upon the autoradiography of axoplasmic transport, in about 1972, signalled the dawn of a new era in neuroanatomy. Certainly it heralded a new burst of activity in connection tracing, a period that is still not over. However, change was already in the wind in other areas of neuroanatomy before 1972. Some of these changes can perhaps be seen as forerunners of the autoradiographic technique. They and others, while hardly causing a revolution like that produced by autoradiography, can be seen as leading up to present-day neuroanatomy's predominant focus on the identification of cell types and their specification in chemical and molecular terms. Improvements in fixation, notably the perfusion of mixed aldehydes~, leading as it did to the wider application of electron microscopy to the central nervous system, were undoubtedly important. Apart from the wealth of new descriptive data on neuronal morphology and on synaptic types and connections that were yielded by electron microscopy3, EM also led to a resurgence of interest in the Golgi Forerunners Walle J. H. Nauta's original method method as a means of classifying the first appeared in 1951 and went through neuronal types present in a particular several transformations, ending in 1967 area of nervous tissue4. From this stems

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the present interest in correlating particular neuron types with their connections, transmitter chemistry, biophysical properties and so on. Perhaps more importantly, however, was the fact that the electron microscope, at that time the major tool in the fledgling science of cell biology, got neuroanatomists thinking about neurons and their supporting cells in cell biological terms. From this has emerged many of our newer neuromorphological research tools, not to mention some fundamental observations on neuronal structure and function. Some of the more obvious contributions of the cell biological school of neuroanatomy- as contrasted with the connectionist school - would be in the areas of the functional morphology of synaptic vesicle release5, the detailed structure of the neuronal cytoskeleton6, and axoplasmic transport as demonstrated by video-enhanced, micro-photography7.

Axol~Lasmictransimrt One of the most fundamental biological properties of neurons is their capacity to transport macromolecules synthesized in the soma along their axon and dendrites. Study of the phenomenon of axoplasmic transport by providing neurons with radioactively labelled amino acid precursors, resulted in the realization that this

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phenomenon could be used for the tracing of connections in the nervous system8'9. The principal advantage of this method over the older Nauta methods was that it obviated the problems caused by 'fibers of passage'. That is, the injected radioactive precursor could only be later fixed in the tissue after having been incorporated first into macromolecules in the cell soma. Injection into axonal bundles traversing the injection site did not lead to spurious connectional patterns because axons lack the capacity for significant protein synthesis. The enormous advantages of this technique were brought home to the neurobiological world in 1972 with the publication of Cowan et al.'s 1° now classic paper on the subject. And by the time the first issue of TINS appeared in 1978, the number of papers published reporting the results of autoradiographically-based, connection-tracing studies must have been in the hundreds. However, by 1978 the popularity of this method (as a means of connection tracing) had already begun to wane. This was because of the rise in popularity of a much less technically demanding method based upon retrograde axoplasmic transport of the enzyme, horseradish peroxidase (HRP) 11. The HRP method, as introduced by LaVail and LaVai112-14 and later modified by several workers 13'1s, provided the opportunity to identify the members of a mixed cell population that gave rise to a particular pathway or set of axon terminal ramifications. This was perhaps its greatest appeal, a type of appeal that is still evident in the use of the more recently introduced retrogradely transported fluorescent dyes 16. However, HRP has essentially come to replace autoradiography as a means of tracing connections anterogradely, especially when conjugated to the lectin, wheat germ agglutinin 17. Certain of the fluorescent dye tracers are also coming to be used in this way (Macchi, G. M., Bentivoglio, M. and Molinari, M., pers. commun.). This is a little surprising when one considers that, used as anterograde tracers, the HRP and fluorescent dye methods suffer from all the disadvantages of 'fibers of passage' as did the older Nauta methods. However, the appeal of direct visualization of axonal profiles, and the instant gratification of looking at them within a day of sacrificing the animal (instead of waiting two weeks or more for an autoradiograph to develop), is evidently too great to resist.

and for numerous brain-gut peptides, has made immunocytochemistry the fundamental neuroanatomical research tool. The immunofluorescence method, in particular, lends itself to visualizing patterns of co-localization of enzymes, transmitters and peptides27 and to the co-localization of these and retrogradely or anterogradely transported dyes2s. Ultimately, the hope would be to specify the transmitter agent of every anatomical pathway. It seems paradoxical, therefore, that most of those long tract connections uncovered in the middle and later 1970s still do not have their transmitters identified. With a few exceptions, notably among the monoaminergic pathways, the immunocytochemical method has told us more about interneurons, especially GABAergic interneurons, than about projection neurons. The other forms of immunocytochemistry in common use are the Cellular and molecular neurobiology avidin-biotin-peroxidase and the If the earlier years of TINS were peroxidase-antiperoxidase methods. dominated by connection tracing in These have the advantage of being neuroanatomy, there are clear signs amenable to electron microscopy but, that the leading edge of the field in to date, they have not lent themselves recent years has a more cell-oriented to combination with other distinct emphasis. If the impetus for connection labels for the localization of two or tracing came from systems physiology, more antigens or of an antigen and a that behind the more recent approach tracer marker in the same tissue. undoubtedly comes from neuro- Several potential second labels using pharmacology and neurochemistry. differently colored chromogens for Several neurobioioglsts in the early HRP or secondary immunoglobulins 1970s had tried to specify the trans- conjugated to ferritin or colloidal gold mitter agents produced by a population (which have potential for electron of neurons or released by their microscopic studies) have been availterminals either by formaldehyde able for some time, but progress in induced fluorescence (for catechol- applying them has been slow. amines) 21, or by identifying autoradioImmunocytochemistry, of course, graphically the presence of a high lends itself to the localization of neural affinity uptake system for a particular antigens other than transmitters, transradiolabelled transmitter22. These prob- mitter-related enzymes of neuropepably represent the earliest forays into tides. The opportunities for structurethe field now often termed 'chemical function correlations are virtually limitneuroanatomy'. However, this form of less in this regard. Numerous examples neuroanatomy truly got off the ground can be noted, including the localization with the application of immunocyto- of region- and neuron-specific phoschemistry to the localization of the phoproteins, many of them associated enzymes involved in catecholamine with the act of synaptic transmission synthesis, tyrosine hydroxylase23 and and the activation of the intracellular dopamine [3-hydroxylase24. Further second messenger systems that succeed momentum came from the immuno- it 29. Even receptors themselves can cytochemical demonstration of certain be recognized by immunocytochemispeptides in the brain and peripheral trya°, and it does not seem unreasonnervous system, particularly the enke- able to consider that the various forms phalins and substance P (Ref. 25) and by of ion channel associated with neuronal the application of receptor binding function will soon also be mapped in techniques to histological sections26. this way - both regionally and in terms Now the ready availability of antisera of single cells. In the area of neural and monoclonal antibodies against development, the expression of cell synthesizing enzymes for other trans- adhesion molecules involved in the mitters, for the transmitters themselves guidance and aggregation of migrating The axoplasmic tracing methods have made many contributions to neurobiology. The greatest achievements are perhaps in two areas. In one, they have undoubtedly created such a synthesis between systems neuroanatomy and systems neurophysiology that it is difficult to conceive of one without the other (see, for example, the contributions of Hubel and WiesellS); this synthesis continues and is being extended to the single cell level as HRP is being injected intracellularly as a means of histologically recovering neurons whose physiological properties have first been determined 19. A second area in which axoplasmic tracing methods have made huge contributions is that of developmental neurobiology, in which the methods have allowed the mapping of many patterns of axon growth and connectional plasticity2°.

504 nerve cells and axons can be tracked 31. The expression and localization of various forms of neuronal cytoskeletal elements6 and of developmentallyand degeneration-regulated macromolecules32 is another area in which immunocytochemistry is adding or will soon add a morphological dimension to the molecular approach. Many of these contributions are taking us away from the expression of neural identity based on the transmitters released and the nature of the immediate act of synaptic transmission, into areas that can be truly regarded as dealing with the fundamental molecular biology of neurons. Although the early hopes of establishing neuronal identity in terms of the expression of uncharacterized antigens discovered by monoclonal antibodies made against crude regional CNS extracts does not seem yet to have realized its early promise33, the hope still remains that certain molecular factors unique to specific sets of neurons will eventually be found. The approach has certainly been useful in permitting clear distinctions to be drawn between different types of neurogiial cell34. Probably the next technical step in specifying cell identity by the expression of transmitters and related substances is already being taken. This is the application of in-situ hybridization, using radiolabelled synthetic oligonucleotide and full-length eDNA probes to detect the presence and abundance of neuron-specificmRNAs. From the limited number of studies that have been done to date, it seems clear that only neurons expressing a particular transmitter-related enzyme or neuropeptide transcribe the relevant part of the genome. Nevertheless, it appears that under certain conditions, neurons not transcribing a particular gene can be induced to do so35. In this are the beginnings of a far more functionally oriented neuromorphology than has previously been possible. The future? Whither neuromorphology in the next 100 issues of TINS? Some obvious predictions seem warranted. First, connection-tracing, except insofar as it is combined with the identification of the transmitter(s) expressed by neurons contributing to a particular pathway or with the analysis of local circuitry, will undoubtedly suffer a further decline in interest. Perhaps there will be a renewed surge of interest as trans-synaptically transported mar-

T I N S - October 1986

kers become more readily available. However, before this field is totally disregarded it would be desirable to see the large mass of data derived from the studies of the 1970s entered into some accessible data base. The problem here will be in making the decision of which data to enter and which to reject as unreliable. Second, multiple labellingstrategies, particularly those involving the colocalization of transmitters, transmitter-related enzymes, other enzymes, peptides, neuron-specific phosphoproteins, receptors, growth factors, and the mRNAs for all these, will undoubtedly come to occupy a prominent place in the next few years. Undoubtedly, also many of these will be used in experimental conditions that show the regulation of expression of one or more of these types of compounds by functional activity36"37. Because many of the methods used for this purpose lend themselves to the study of human post-mortem brains, and because of the growing interest in the chemical bases of many neurological diseases of humans, one can expect to see a growing number of these studies devoted to man. Third, techniques for analysing histologically characterized neurons at the electron microscope level, particularly in serial section analysis, have been available for some time38. Work will undoubtedly continue in this area as more intracellular injection and immunocytoehemicalstudies are done, and especially as double-labellingstrategies for combining the two become more amenable to electron microscopy. However, it is doubtful that more than a handful of neuroscientists will be inclined to expend the great deal of time that this kind of work necessitates and, even among these, still fewer will have sufficient dedication to study more than a token sample of cells. Computerized reconstruction systems, though available for some years, and enthusiastically heralded, have yet to make significant strides in alleviating this problem. Fourth, there is bound to be a continuing period during which novel markers for neuronal identity, such as surface glycoconjugates, intracellular enzymes, uncharacterized antigens identified by monoclonal antibodies, specific phosphoproteins, substrates for second messengers, etc., will continue to be sought. Let us hope that the end result of this period will be a full characterization of neuronal identities

in a manner that goes beyond their transmitter specificity and connectional relationships and which tells us something about their functional specificity and why they express a particular morphology. Perhaps a map of a specific neuronal class, showing the distribution of its receptors and channels in its membrane, the substrates within its cytoplasm necessary for receptor-mediated biochemical processes, the trophic agents it needs to keep it alive, the transmitter that it secretes, the exact molecular composition of its cytoskeletal elements, etc., is not too distant a hope for the future. Let us hope that these studies are accompanied by significant quantification of the cell populations, something that has been sadly lacking up to this point. For the future, one senses that there may be a greater emphasis on the direct visualization of living processes in single nerve cells or in neuronal (and neuroglial) populations. In a sense the 2-deoxy-D-glucose method and the histochemical staining for metabolic enzymes such as cytochrome oxidase, represented first steps in this direction, although the time resolution of deoxyglucose and the unresolved difficulty of bringing its spatial resolution down to the single cell level has limited its applicability. The use of PET scanning in experimental animals has been similarly retricted by its limited spatial resolution. However, in the human brain at least, there are promising signs that PET scanning may be used to visualize receptor localizations very effectively 39. MRI and related methods also await application to experimental conditions. With the application of phosphorous spectroscopy to brain imaging, it may be possible to extend the range of applicability to many additional neural processes. Possibly the most promising methods for directly visualizing living processes in real time in experimental animals are those that make use of video-enhanced microscopy, and voltage sensitive dyes. Both have already yielded significant results in some diverse areas of application7'4°-42. Undoubtedly they will be used even more widely in the future, probably both at the level of the single cell and of large, functionallyinter-related neuronal populations. Much that I have discussed presupposes that the major forms of neuromorphological research will still be conducted on experimental animals. It is doubtful that it could be otherwise.

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Although much neuromorphology has a more functional orientation than it ever had, neuronal form and connectivity, like neuronal function, only reach their full expression in the living, intact animal. The current climate of opinion regarding animal research cannot change this. M e t h o d s are becoming available for maintaining large masses of neural tissue 43, or even whole brains, in vitro for long periods, and while they are attracting morphological studies, they cannot as yet substitute for studies in the animal itself. Many now speak of a revolution that has occurred in neuroanatomical methodology, and certainly in recent years there has b e e n a tremendous increase in the range of morphological techniques available to ~the neuroscientisL~These have in turn generated a great deal of interest in neuronal structure and connectivity, especially as these relate to function. Technique is perhaps more important in neurobiology than in many o t h e r areas of biological science, because so few of the methods can be standardized. W h e n asked why his ablation experiments always worked better than those of others, the physiologist Flourens was fond of saying 'technique is everything', something which backfired o n him when Hitzig used the same expression to explain why he, Hitzig, could elicit m o v e m e n t s by stimulating the m o t o r cortex and Flourens could not. But technique for its own sake is of little importance. As the pathologist Virchow said ' m a n y anatomists concern themselves all too frequently with unnecessary measurements and meaningless counts'. Technique is only important insofar as it enables us to answer questions. Let us hope that the new wave of neuromorphological technology will help us continue to address more and more fundamental issues in neurobiology for, as Floyd Bloom once put it 'the gains in brain are mainly in the stain'. Selected references 1 Nanta, W. J. H. and Ebbesson, S. O. E., eds (1970) Contemporary Research Methods in Neuroanatomy, Springer Verlag 2 Karnovsky, M. J. (1965)./. CellBioL 27,137A 3 Peters, A., Palay, S. L. and Webster, H. de F. (1976) The Fine Structure o f the Nervous System (2nd edn), Sannders 4 Jones, E. G. (1975) J. Comp. NeuroL 160, 205-268 5 Heuser, J. E., Reese, T.S. and Landis, D. M. (1974)J. Neurocytol. 3, 109-122 6 Hirokawa, N. (1986) Trends Neurosci. 9, 67-71 7 Schnapp, B. J. and Rcese, T.S. (1986)

Trends Neurosci. 9, 155-162 8 Lasek, R. J., Joseph, B. S. and Whitlock, D. G. (1968) Brain Res. 8, 319-336 9 Hendrickson, A. E. (1969) Science 165, 194-196 10 Cowan, W. M., Gottlieb, D. I., Hendrickson, A. E., Price, J. L. and Woolsey, T. A. (1972) Brain Res. 37, 21-51 11 Kristensson, K. and Olsson, Y. (1971) Brain Res. 29, 363-365 12 LaVail, J. H. and ].,¢Vail, M.M. (1972) Science 176, 1417-1417 13 DeOlmos, J., Hardy, H. and Helmet, L. (1978) J. Comp. Neurol. 181,213-244 14 LaVail, J. H., Winston, K. R. and Tish, A. (1973) Brain Res. 58,470-477 15 Mesulam, M-M. (1978) J. Histochem. Cytochem. 26, 106-117 16 Kuypcrs, H. G. J. M., Catsman-Berrevocts, C. E. and Padt, R. E. (1977) Neurosci. Lett. 6, 127-135 17 Schwab, M. E., Javoy-Agid, F. and Agid, Y. (1978) Brain Res. 152, 145-150 18 Hubel, D. M. and Wiesei, T. N. (1977)Proc. R. Soc. London Set. B. 198, 1-59 19 Yen, C-T., Conley, M., Hendry, S. H. C. and Jones, E.G. (1985) J. Neurosci. 5, 2254-2268 20 Rakic, P. (1981) Science 214, 928-931 21 Falck, B., Hillarp, N. A., Thieme, G. and Torp, A. (1962)I. Histochem. Cytochem. 10, 348-354 22 H6kfelt, T. and Ljiingdahl, A.L. (1972) Exp. Brain Res. 14, 354-362 23 Pickel, V. M., Job, T. H., Field, P. M., Becker, C.G. and Reis, D.J. (1975) J. Histochem. Cytochem. 23, 1-12 24 Hartman, B. K., Zide, D. and Udenfriend, S. (1972) Proc. Natl Acad. Sci. USA 69, 2722-2726 25 Snyder, S. H. (1980) Science 209, 976-983 26 Kuhar, M. J. and Yamamura, H. I. (1976) Brain. Res. 110, 229-243

27 Jones, E. G. and Hendry, S. H. C. (1986) Trends Neurosci. 9, 71-76 28 Sawchenko,P. E. and Swanson,L. W. (1981) Brain Res. 210, 31-52 29 Nestler, E. J. and Greengard, "P. (1984) Protein Phosphorylation in the Nervous System, John Wiley & Sons

30 Schoch, P., Richards, J.G., Hating, P., Takacs, B., St~hli, C., Staehelin, W., Haefely, W. and M6hler, H. (1985) Nature 314, 168-171 31 Edelman, G. M. (1985)Annu. Rev. Biochem. 54, 135-171 32 Skene, J. H. P. and Willard, M. (1981) J. Neurosci. 1,419-426 33 Zipser, B. and MacKay, R. (1981) Nature 289, 549-552 34 Raft, M. C., Abney, E.R., Cohen, J., Lindsay, R. and Noble, M. (1981)J. Neurosci. 3, 1289-1298 35 Wolfson, B., Manning, R. W., Davis, L. G., Arentzen, R. and Baldino, F., Jr (1985) Nature 315, 59-61 36 Hendry, S. H. C. and Jones, E. G. (1986) Nature 320, 750-753 37 Hendry, S. H. C. and Kennedy, M. B. (1986) Proc. Natl Acad. Sci. USA 83, 1536-1540 38 Fair6n, A., Peters, A. and Saldanha, J. (1977) J. Neurocytol. 6, 311-337 39 Stahl, S. M., Le~nders, K. L. and Bowery, N. G. (1986) Trends Neurosci. 9, 241-245 40 Purves, D., Hadley, R. D. and Voyvodic, J. T. (1986) J. Neurosci. 6, 1051-1060 41 Lichtman, J. W., Wilkinson, R. S. and Rich, M. M. (1985) Nature 314, 357-359 42 Blasdel, G. G. and Salama, G. (1986)Nature 321,579-584 43 Gahwiler, B. H. (1981)J. Neurosci. Meth. 4, 329-342 Edward G. Jones is at the Department o f Anatomy and Neurobiology, University of California, Irvine, CA 92717, USA.

The neural mechanisms of cognitive functions can now be studied directly Vernon B. Mountcastle The general theme of this essay is that progress in the brain and psychological sciences in the last decades has provided a strong base from which to plan research aimed at the long-range goal of neuroscientists; to understand the neural mechanisms of the higher functions of the brain, those commonly labelled cognitive. These programs will initially address intermediate-level questions, for example: how primates perceive the world around them and store and later access information about it; how they plan and carry out actions upon the external environment; how they attend to one sensory channel to the exclusion of others; and how they

learn to modify behavior in the light of past experience. No answers for these or similar questions are to be found in the neurobiology of simple brains. The accelerating acquisition of knowledge concerning the structure and function of the brain combines with three other developments to create a unique opportunity in the history of brain science. The first is a change in the general concept of the functional organization of the brain. The second is the development of a field of neural modelling and computation inspired and constrained by the functional properties of real neurons and neural ensembles 1-5. The third is new insight

~) 1986.EbevierSciencePublishersB.V..Amsterdam 0378- 5912/86/$02.00