PATHWAYS OF DISCOVERY

Neuroscience:Breaking Down Scientific Barriersto the Study of Brain and Mind

Eric R. Kandel and Larry R. Squire

During tlic latter part of the 20th century,, the study of the brain moved from a peripheral positionwithin both the biological and psychological sciences to become an interdisciplinary field called neu-roscience that now occupies a central position within each discipline. This realignment occurred be-cause the biological study of the brain became incorporated inlo a common framework with ceil andmolecular biology on the one side and with psychology on the other. Within this new framework, thescope of neuroscience ranges from genes to cognition, from molecules to mind.

What led to the gradual incorporation of neuroscience into the centralcore of biology and to its alignment with psychology? From the

perspective of biology at the beginning of the 20th century, thetask of neuroscience—to understand how the brain developsand then functions to perceive, think, move, and remember—seemed impossibly difficult. In addition, an intellecmal barri-

er separated neuroscience from biology, because the lan-guage of neuroseience was based more on neuroanatomyand electrophysiology than on the universal biologicallanguage of biochemistry. During the last 2 decades thisbarrier has been largely removed. A molecular neuro-seience became established by focusing on simple sys-tems where anatomy and physiology were tractable. As aresult, neuroseience helped delineate a general plan for

neural cell function in whieh the cells of the ner\'ous sys-tem are understood to be governed by variations on uni-

versal biological themes.

From the perspective of psychology, a neural approach tomental processes seemed too reductionistic to do justice tothe complexity of cognition. Substantial progress was re-quired to demonstrate that some of these reductionist goalswere achievable within a psychologically meaningful frame-work. The work olVemon Mountcastle, David Hubci. Torsten

Wiesel, and Brenda Milner in the 1950s and 1960s, and the advent of brain Imaging in the 1980s,showed what could be achieved for sensory processing, perception, and memory. As a result of theseadvances, the view gradually developed that only by exploring the brain could psychologists fullysatisfy their interest in the cognitive processes that intervene between stimulus and response.

Here, we consider several developments that have been partieularly important for the maturationof neuroseience and for the restmcturing of its relationship to biology and psychology.

The Emergence of a Cellular and Molecular NeuroseienceThe modern cellular science of the nervous system was founded on two important advances: theneuron doctrine and the ionic hypothesis. The neuron doctrine was established by the brilliantSpanish anatomist Santiago Ramón y Cajal (1). who showed that the brain is composed of discretecells, called neurons, and that these likely serve as elementary signaling units. Cajal also ad-vanced the principle of connection specificity, the central tenet of which is that neurons formhighly specific connections with one another and that these connections are invariant and definingfor each species. Finally, Cajal developed the principle of dynamic polarization, according towhich information flows in only one direction within a neuron., usually from the dendrites (theneuron's input component) down the axon shaft to the axon tenninals {the output component). Al-though exceptions to this principle have emerged, it has proved extremely influential, beeause ittied structure to function and provided guidelines for eonstrueting circuits from the images pro-vided in histological sections of the brain.

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Artful brain. Cross anatomy madebeautiful in a Christopher Wren illus-tration from a 1664 anatomy text.

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A Timeline ofNeuroscience

2nd CenturyA.D.Galen of Perga-mum identifiesthe brain as theorgan of themind.

17th CenturyThe brain be-comes acceptedas tKe substrateof mental liferather than Itsventricles, asearty writershad proposed.

1664Thomas Willispublishes Cerebrianatome, with it-lustrations of thebrain by Christo-pher Wren. It isthe most com-prehensive trea-tise on brainanatomy andfunction

^published up tothat time.

1791Luigi Calvani re-veals the electricnature of nervousaction by stimu-lating nerves andmuscles of frogtegs.

1808Franz Joseph Gallproposes thatspecific brainregions controlspecific func-

> tions.

I 1852[ Hermann vonf Helmholtimea-• sures the speedl o f a nerve im-^ fHjbe in the frog.

Í 1879: Wilhelm Wundt' establishes the' first laboratory* of experimentalf psychology in

' [, Germany,

Cajal and his contemporary Charles Sherrington (2) fur-ther proposed that neurons contact one another only at spe-cialized points called synapses, the sites where one neuron'sprocesses contact and communicate with another neuron. Wenow know that at most synapses, there is a gap of 20 nm—the synaptic cleft—between the pre- and postsynaptic cell. Inthe 1930s, Otto Loewi. Henry Dale, and Wilhelm Feldbergestablished (at peripheral neuromuscular and autonomiesynapses) that the signal that bridges the synaptic cleñ is usu-ally a small chemical, or neurotransmitter. which is releasedfrom the presynaptic temiinal, diffuses across the gap. andbinds to receptors on the postsynaptic target cell. Dependingon the specific receptor, the postsynaptic cell can either beexcited or iniiibited. It totik some time to establish that chem-ical transmission also occurs in the central nervous system,but by the 1950s the idea had become widely accepted.

Even early in the 20th century, it was already understoodthat nerve cells have an electrical potential, the resting mem-brane potential, across their membrane, and that signalingalong the axon is conveyed by a prop-agated electrical signal, the action po-tential, which was thought to nullifythe resting potential. In 1937 AlanHodgkin discovered that the actionpotential gives rise to local currentHow on its advancing edge and ihatthis current depolarizes the adjacentregion of the axonal membrane suffi-ciently to trigger a traveling wave ofdepolarization. In 1939 Hodgkin and.'Andrew Huxley made the surprisingdiscovery that the action potentialmore than nullifies the resting poten-tial—it reverses it. Then, in the late1940s, Hodgkin, Haxley. and BernardKatz explained the resting potentialand the action potential in terms ofthe movement of specific ions—potassium (K" ), sodium (Na*). andehloride (Cl)—through pores (ionchannels) in the axonal membrane.This ionic hypothesis unified a targebody of descriptive data and offered the first realistic promisethat the nervous system could be understood in terms ofphysicoehemical principles common to all of cell biology (3).

The next breakthrough came when Katz. Paul Fatt. andJohn Eccles showed that ion channels are also ftindamental tosignal transmission across the synapse. However, rather thanbeing gated by voltage like the Na"" and K* channels criticalfor action potentials, excitatory synaptic ion channels aie gat-ed chemically by ligands such as the transmittei- acetylcholine.During the 1960s and 1970s, neuroseientists identified manyiunino acids, peptides. and otlier small molecules as chemicaltransmitters, including acetylcholine, glutamate. GABA,glycine, serotonin, dopamine. and norepinephrine. On the or-der of 100 chemical transmitters have been discovered to date.In the 1970s, some synapses were found to release a peptidecotmnsmittcr that can modify the action of the classic, small-moleeule transmitters. Tlie discovery of chemical neurotrans-mission was followed by the remarkable discovery that trans-mission between neurons is sometimes electrical (4). Electri-cal synapses have smaller synaptic eiefts. which are bridgedby gap junctions and allow cunent to flow between neurons.

In the late 1960s information began to become available¡ibout the biophysical and biochemical structure of ionic

Seeing neurons. Anatomist Ramón y Cajalused Colgi's stain to examine individualnerve cells and their processes,

pores and the biophysical basis for their selectivity and gat-ing^how they open and close. For example, transmitterbinding sites and their ion channels were found to be em-bodied within different domains of multimeric proteins. Ionchannel selectivity was found to depend on physical-chemical interaction between the channel and the ion. andchannel gating was found to result from conformationalchanges within the channel (5).

The study of ion channels changed radically with the de-velopment of the pateh-clamp method in 1976 by Erwin Ne-her and Bert Sakmann (6), which enabled measurement ofthe current flowing through a single ion channel. This pow-erful advance set the stage for the analysis of ehannels at themolecular level and for the analysis of functional and con-

formational change in a singlemembrane protein. When appliedto non-neuronal cells, the methodalso revealed that all cells—evenbacteria—express remarkably sim-ilar ion channels. Thus, neuronalsignaling proved to be a specialcase ofa signaling capability in-herent in most cells.

The development of patchclamping coincided with the ad-

vent of molecular cloning, and Ihesctwo methods brought neuroscientistsnew ideas based on the first reports ofthe amino acid sequences of ligand- andvoltage-gated channels. One of the keyinsights to emerge from molecularcloning was that amino acid sequencescontain clues about how receptor pro-teins and voltage-gated ion channel pro-teins are arranged across the cell mem-brane. The sequenee data also oftenpointed to unexpected structural rela-tionships (homoiogies) among proteins.These insights, in turn, revealed similar-ities between molecules found in quitedifferent neuronal and non-neuronalcontexts, suggesting that they may serve

similar biological flinctions.

By the early 1980s, it became clear that synaptic ac-tions were not always mediated directly by ion channels.Besides ionotropic receptors, in which ligand binding di-rectly gates an ion channel, a second class of receptors, themetabotropic receptors, was discovered, Here the bindingof the ligand initiates intracelluiar metabolic events andleads only indirectly, by way of "second messengers," tothe gating of ion channels (7).

The cloning of fnetabotropic receptors revealed that man\of them have seven membrane-spiinning regions and are ho-mologous to bacterial rhodopsin as well as to the photorecep-tor pigment of organisms ranging from fruit flies to humans.Further, the recent cloning of reecptors for the sense of smell(S) revealed that at least 1000 metabotnipic receptors are ex-pressed in the mammalian olfactory epithelium and that sim-ilar receptors are present in flies and worms. Thus, it was in-stantly understood thai tlie class of receptors used for photo-transduction, the initial step in visual perception, is also usedfor smell and aspects of taste, and that these receptors sharekey features with many other brain receptors that workthrough second-messenger signaling. These discoveriesdemonstrated the evolutionary conservation of receptors and

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emphasized the wisdom of studying a wide variety of experi-mental systems^—vertehrates. invertebrates, even single-celled organisms-^o identify broad biologieal prineiples.

The seven transmembrane-spanning receptors activateion channels indirectly through coupling proteins (G pro-teins). Some G proteins have been found lo activate ionchannels directly. However, the majority of G proteins acti-vate membrane enzymes that alter the level of second mes-sengers, such as cAMP, cGMP, or inositol triphosphate,wliich initiate complex intracellutar events leading to the ac-tivation of protein kinases and phosphatases and then to themodulation of channel permeability, receptor sensitivity; andtransmitter release. Neuroscientists now appreciate thatmany ol" these synaptic actions are mediated intraceilularlyby protein phosphorylation or dephosphorylation (9), Nervecells use such covalent modifications to control protein ac-tivity reversibly and thereby to regulate function. Phospho-rylation is also eritical in other cells for the action of hor-mones and growth factors, and for many other processes.

Directly controlled synaptic actions are fast, lasting mil-liseconds, but seeond-messenger actions last seconds to min-utes. An even slower synaptic action, lasting days or more,has been found to be important for long-tenii memory. Inthis case, protein kinases activated by seeond messengerstranslocate to the nucleus, where they phosphorylate tran-scription factors that alter gene expression, initiate growtli ofneuronal processes, and inerease synaptic strength.

Transmitter molecules bind10 excitatory receplors. receptorchannels open, and Na* enters

tfie postsynaptic cetl

The synapse. A presynaptic neuron propagates a signal by releasing neurotrans-mitter molecules that diffuse across the synaptic cleft to bind to receptors on thepostsynaptic cell.

Ionotropic and metabotropic receptors have helped toexplain the postsynaptic side of synaptie transmission. Inthe 1950s and 1960s. Katz and his colleagues turned to thepresynaptic terminals and discovered that chemical trans-mitters, such as acetylcholine. are released not as singlemolecules but as packets of about 5000 molecules calledquanta (10). Each quantum is packaged in a synaptic vesi-cle and released by exocytosis at sites called active zones.The key signal that triggers this sequenee is the influx ofCa"' with the action potential.

In recent years, many proteins involved in transmitter re-lease have been identified (II). Their functions range (Vomtargeting vesicles to active zones, tethering vesicles to thecell membrane, and fusing vesicles with the cell membraneso that their contents can be released by exocytosis. Thesemoieeular studies reflect another example of evolutionaryconservation: The molecules used for vesicle fusion andexocytosis at nerve terminals are variants of those used forvesicle fusion and exocytosis in all cells.

A Mechanistic View of Brain DevelopmentThe discoveries of molecular neuroscience have dramatical-ly improved the understanding of how the brain develops itscomplexity. The modern moleeular era of developmentalneuroscience began when Rita Levi-Montai ein i and StanleyCohen isolated nerve growth factor (NGF). the first peptidegrowth factor to be identified in the ner\'ous system (12).They showed that injection of antibodies to NGF into new-bom mice caused the death of neurons in sympathetic gan-glia and also reduced the number of sensory ganglion cells.Thus, the survival of both sympathetic and sensory neuronsdepends on NGF. Indeed, many neurons depend for theirsurvival on NGF or related molecules, which typically pro-vide feedback signals to the neurons from their targets. Suchsignals are important for programmed cell death—apopto-sis—a developmental strategy which has now proved to beof general importance, whereby many more ceUs are gener-ated than eventually survive to become functional units withprecise connectivity. In a major advance, genetic study ofworms has revealed the ced genes and with them a universalcascade critical for apoptosis in which proteases—the cas-pases—are the final agents for cell death (13).

Cajai pointed out the extraordinary precision of neuronalconnections. Tlie first compelling insights into how neuronsdevelop their preeise connectivity came from Roger Sperry'sstudies of the visual system of frogs and salamanders begin-ning in the 1940s, which suggested that axon outgrowth is

guided by molecular cues. Sjierry s key find-ing was that when the nerves from the eyeare cut, axons find their way back to theiroriginal targets. These seminal studies ledSperry in 1963 to formulate the chemoafFin-ity hypothesis (14).. the idea that neuronsform connections with their targets based ondistinctive and matching molecular identitiesthat they acquire early in development.

Stimulated by these early contributions,molecular biology has radically trans-formed the study of nervous system de-velopment from a descriptive to a mecha-nistic field. Three genetic systems, theworm Caenorhabditis elegam. the fruit flyDrosophila melanoga.^ter. and the mouse,have been invaluable; some ofthe mol-ecules for key developmental steps in themouse were first characterized by genetic

screens in worms and flies. In some cases, identicalmolecules were found to play an equivalent role throughoutphylogeny. The result of this work is that neuroscientistshave achieved in broad outline an understanding ofthemolecular basis of nervous system development (15). Arange of key molecules has been identified, including spe-cific inducers. morphogens, and guidance molecules impor-tant for differentiation, process outgrowth, pathfinding. andsynapse formation. For example, in the spinal cord, neuronsaehieve their identities and characteristic positions largelythrough two classes of inductive signaling moleeules oftheHedgehog and bone morphogenic protein families. Thesetwo groups of molecules control neuronal differentiation inthe ventral and dorsal halves ofthe spinal cord, respeetively.and maintain this division of labor through most ofthe ros-trocaudal length ofthe nen'ous system.

The process of neuronal pathfinding is mediated byboth short-range and long-range cues. An axons growthcone can encounter cell surface eues that either attract or

1891Withelm vonWaldeyer-Hartzintroduces theterm neuron.

1897ChartesSherringtonintroduces theterm synapse.

1898-1903Edward Thorndikeand Ivan Pavlovdescribe opérantand classicalconditioning, twofundamentaltypes of teaming.

1906Santiago Ramóny Cajat sgmma-riies compeltingevidence for theneuron doctrine,that the nervoussystem is com-

Alois Aliheimefdescritas thepathotogy of theneurodegenera-tive disease thatcomes to bear his

1914Henry Daledemonstratesthe physiologicataction of acetyl-choline, which islater identifiedas a neuro-transmitter.

1929In a famousprogram of lesionexperiments inrats. Karl Lashleyattempts tolocalize memoryin the brain.

Hans Berger uséshuman scatpelectrodes todemonstrateetectroen-cephatography.

1928-32Edgar Adriandescribes methodfor recordingfrom singlesensory andmotor axons;H. Keffer Harttineapplies thismethod to therecording ofsingle-cetl activ-ity in the eye ofthe ho^se5hoe^ -crab. '!

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1940sAlan Hodgkin,Andrew Huxley,and Eternard Katz

«Kpiain eiKtricalactivity of neu-rons by concen-tration gradientsof ions and n^ove-ment of ions"" " " ugh pores.

th Cole de-s th« volt-

,*'-clamp tech-nique to measurecurrent flowacross the cell

" jibrane.

' Donald Hebb in-troduces a synap-tlc [earning rute,which becomesknown as theHebb rute.

19305 t o 19505The chemical na-ture of synapticiransmission is es-tablished by OttoLoewi. Henry Dale,Wilhelm Feldberg,Stephen Kuffler,and Bernard Katzat periphieralsynapses and isextended to thespinal cord by JohnEccles and others

Wilder Penfieldand TheodoreRasmussen mapthe motor andsensory ho-munculus and il-lustrate localiza-tion of function inthe human brain.

repel it. For example, ephrins are membrane-bound, aredistributed in graded fashion in many regions of the ner-vous system, and can repel growing axons. Other cues,such as Uie netrins and the semaphorins, arc secreted indiffusible form and act as long-range chemoattractants orehemorepellents. Growth cones ean also reaet to the samecues diiferently at different developmental phases, for ex-ample, when crossing the midline or when switching frompathfinding to synapse formation. Finally, a large numberof molecules are involved in synapse formation itself.Some, such as neuregulin, erbB kinases, agrin, and MuSK.organize the assembly of the postsynaptic machinery,whereas others, such as the laminins, help to organize thepresynaptic differentiation of the active zone.

These molecular signals direct differentiation, migration,process outgrowth, and synapse fonnation in the absence ofneural activity. Neural activity is needed however, to refinethe connections further so as to forge the adult pattern ofconnectivity (¡6). The neural activity may be generatedspontaneously, especially early in development, but later de-pends importantly on sensory input. In this way. intrinsic ac-tivity or sensory and motor experienee can lielp specify aprecise set of functional connections.

The Impact of Neuroscience on Neurology and PsychiatryMolecular neiuoscience has also reaped substantial benefitsfor clinical medicine. To begin with, recent advances in thestudy of neural development have identified stem cells, bothembryonic and adult, which offerpromise in cell replacement ther-apy in Parkinson's disease, de-myelinating diseases, and otherconditions. Similarly, new in-sights into axon guidancemolecules offer hope for nerveregeneration after spinal cord in-jury. Finally, because most neuro-logical diseases are associatedwitlî ceil death, the discovery inworms of a universal genetic pro-gram for cell death opens up ap-proaches fbr cell rescue based on,for example, inhibition of thecaspase proteases.

Next, consider the impact ofmolecular genetics. Hunting-ton's disease is an autosomaldominant disease marked byprogressive motor and cognitiveimpairment that ordinarilymanifests itself in middle age. The major pathology is eelldeath in the basal ganglia. In 1993, the Huntington's Dis-ease Collaborative Research Group isolated the gene re-sponsible for the disease (17). It is marked by an extendedseries of trinucleotide CAG (eytosine. adenine. guanine)repeats, thereby placing Huntington's disease in a newclass of neurological disorders—the trinucleotide repeatdiseases—that now constitute the largest group of domi-nantly transmitted neurological diseases.

The molecular genetic analysis of more complex degener-ative disorders has proceeded more slowly. Still, three genesassociated with familial Alzheimer's disease—those thatcode for the amyloid precursor protein, presenilin I. and pre-senilin 2^havc been identified. Molecular genetic studieshave also identified the first genes that modulate the severity

and risk of a degenerative disease (¡8). One alíele (APO E4)is a significant risk factor for late-onset Alzheimers disease.Conversely, the APO E2 alíele may actually be protective. Asecond risk factor is a^-macroglobulin. All the Alzheimer's-related genes so far identified participate in eitlier generatingor scavenging a protein (the amyloid peplide), which is toxicat elevated levels. Smdies directed at this peptide may lead toways to prevent the disease or halt its progression. Similarl>.the discovery of [i-secrctase and perhaps y-secretase, theetizymes involved in the processing of ß amyloid, representdramatie advances that may also lead to new treamients.

With psychiatric disorders, progress has been slower fortwo reasons. First, diseases such as schizophrenia, depres-sion, obsessive eompulsive disorders, anxiety states, anddrug abuse tend to be complex, polygenic disorders that aresignificantly modulated by environmental factors. Second incontrast to neurological disorders, little is known about theanatomical substrates of most psychiatric diseases. Given thedifficulty of penetrating the deep biology of mental illness, itis nevertheless remarkable how much progress has beenmade during the past 3 deeades (19). Arvid Carlsson ;mdJulius Axelrod carried out pioneering studies of biogenicamines, which laid the foundation for psychopharmacology,and Seymour Kety pioneered the genetic study of mental ill-ness (20). Currently, new approaches to many conditions,such as sleep disorders, eating disorders, and drug abuse., areemerging as the result of insights into tlie cellular and molec-ular machinery that regulates specific behaviors (2¡). More-

over, improvements in diagnosis, the bet-ter delineation of genetic contributions topsychiatric illness (based on twin andadoption studies as well as studies of af-fected families), and the diseovery of spe-cific medications for treating schizophre-nia, depression, and anxiety states havetransformed psychiatry into a therapeuti-eally effeetive medical specialty that isnow closely aligned with neuroscience.

Little man inside. This 1950 homunculus sum-marized studies of the cerebral localization ofmotor function.

A New Alignment of Neuroscience andPsychological ScienceThe brain's computational power is con-ferred by interactions among billions ofnerve cells, which are assembled into net-works or circuits that cany out specificoperations in support of behavior andcognition. Whereas the molecular ma-chinery and electrical signaling propertiesof neurons are widely conserved aerossanimal species, what distinguishes one

species from another, with respect to their cognitive abilities,is the number of neurons and the details of their connectivity.

Beginning in the 19th century there was great interest inhow these cognitive abilities might be localized in the brain.One view, first championed by Franz Joseph Gall, was thatthe brain is eomposed of specialized parts and thai aspectsof perception, emotion, and language can be locaiized toanatomically distinct neural systems. Another view, champi-oned by Jean-Pierre-Marie Flourens. was that cognitivefunctions are global properties ari.sing from the integratedaetivity of the entire brain. In a sense, the history of neuro-science can be seen as a gradual ascendancy of the localiza-tionist view.

To a large extent, the emergence of the localizationistview was built on a eentury-old legacy of psychological sci-

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cnce. When psychology emerged as an experimental sciencein the late mtli century, its founders, Gustav Fechner andWilhelm Wundt. focused on psychophysics—the quantita-tive relationship between physical stimuli and subjectivesensation. The success of this endeavor encouraged psychol-ogists to study more complex behavior, which led to a rigor-ous, laboratory-based tradition termed behaviorism.

Led by John Watson and later by B. F. Skiimer, behavior-ists argued that psychology should be concerned only withobservable stitïiuli and responses, not with unobservableprocesses that intervene between stimulus and response.This tradition yielded lawful principles of behavior andlearning, but it proved limiting. In the 1960s, behaviorismgave way to a broader approach concerned with cognitiveprocesses and internal representations. This new emphasisfocused on precisely those aspeets ot" mental life—from per-ception to action^that had long been of interest to neurolo-gists and other students of the nervous system.

The first cellular studies of brain systems in the 1950s il-lustrated dramatically how much neuroscience derived frompsychology and conversely how much psychology could, inturn, inform neuroscience. In using a cellular approach, neu-roscientists relied on the rigorous experimental methods ofpsychophysics and behaviorism to explore how a sensorystimulus resulted in a neuronal response. In so doing, theyfound cellular support for localization of ftinclion: Differentbrain regions had difierent cellular response properties. Thus.it became possible in the study of behavior and cognition tomove beyond description to an exploration of the meehanismsunderlying the internal representation of the extemal world.

In the late 1950s and 196ns Mountcastle. Hubel. andWiesel began using cellular approaches to analyze sensoryprocessing in the cerebral coriex of cats and monkeys (22).Their work provided the most fundamental advance in under-standing the organization of the brain since the work of Cajalat the turn of the century. The cellular physiological tech-niques revealed that the bniin both i'ilters and transfomis sen-sory information on its way to and within the cortex, and thatthese transfomiations are critical for perception. Sensory sys-lems analyze, decompose, and then restructure raw sensoryinformation according to built-in connections and rules.

Mountcastle found Iliat single nerve cells in the primary so-matic sensory cortex respond to st-ieeific kinds of touch; Somerespond to superficial touch iind others to deep pressure, butcells almost never respond to btith. Tlie dißerent ceU types aresegregated in vertical columns, which comprise thousands ofneurons and extend about 2 mm ftom the cortical surface tothe wliite tnatter below it. Mountcastle proposed that each col-umn serves as an integrating unit, or logical mLxiule. and tliatthese columns are the basic mode of cortical organization.

Single-cell recording was pioneered by Edgar Adrian andapplied to the visual system of invertebmtes by M. Keffer Hart-line and to the visual system of mammals by Stephen KufFler,the mentor of Mubel and Wiesel. In recordings from the retina,Kuffler discovered that, rather tlian signaling absolute levels oflight, neurons signal contrast between spots of light and dark.hi the visual cortex, Hubel and Wiesel found that most cells nolonger respond to spots of light. For example, in ana VI at theoccipital pole of the cortex, neurons respond to specific visualfeatures such as lines or bars in a particular orientation. More-over, eells witli similar orientation preferences were found togroup together in vertical colmnns similar to those that Mount-castle had found in sotnatosensory cortex. Indeed an indepen-dent system of vertical columns—the ocular dominancecolumns—was found to segregate infonnation arriving from

the two eyes. These nsults provided an entirely new view ofthe anatomical organization of the cerebral cortex.

Wiesel and Hubel also investigated the effects of early sen-sory deprivation on newborn animals. They found that visualdeprivation in one eye profoundly alters the organization ofoeular dominance columns (23). Columns receiving inputfrom the closed eye shrink, and those receiving input from theopen eye expand. These studies led to the dlscover> that eyeclosure alters the pattem of synchronous activity in the twoeyes and that tliis neural activity is essential for fine-tuningsynaptic connections during visual system development (16).

In the extrastriate cortex beyond area VI, continuingelectrophysiological and anatomical studies have identifiedmore than 30 distinct areas important for vision (24). Fur-ther, visual information was found to be analyzed by twoparallel processing streams (25). The dorsal stream, con-cerned with where objects are located in space and how toreach objects, extends írom area VI to the parietal cortex.The ventral stream extends from area VI to the inferior tem-poral cortex and is concerned with analyzing the visual formand quality of objects. Thus, even the apparently simple taskof perceiving an object in space engages a disparate collec-tion of specialized neural areas that represent different as-pects of the visual information—what the object is. where itis located and how to reach for it.

A Neuroscience of CognitionThe initial studies of the visual system were perfomied inanaesthetized cats, an experimental preparation far removedfrom the behaving and thinking human beings that are thefocus of interest for cognitive psychologists. A pivotal ad-vance occurred in the late 1960s when single-neuron record-ings were obtained from awake, behaving monkeys that hadbeen trained to perform sensory or motor tasks (26). Witiithese methods, the response of neurons in the posterior pari-etal cortex to a visual stimulus was found to be enhancedwhen the animal moved its eyes to attend to the stimulus.This moved the neurophysiological study of single neuronsbeyond sensory processing and showed that reductionist ap-proaches could be applied to higher order psychological pro-cesses such as selective attention.

It is possible to correlate neuronal firing with perceptionrather direetiy. Thus, building on earlier work by Mountcas-tle. a monkey's ability to discriminate motion was found toclosely match the performance of individual neurons in areaMT, a cortical area concerned with visual motion processing.Further, electrical microstimulation oi' small clusters of neu-rons in MT shins the monkey's motion judgments toward fhedirection of motion that the stimulated neurons prefer (27).Thus, activity in area MT appears sufficient for the percep-tion of motion and for initiating perceptual decisions.

1950sKarl von Frisch,Konrdd Loreni,and NikolaasTinbergenestablish thescience ofethology (animalbehavior in natu-rat contexts) andlay the founda-tion for neiiro-et hoi ogy.

1955-60Vernon Mount-castle, DavidHübet, andTorsten Wieselpioneer single-cell recordingfrom mammaliansensory cortex;NilS'Ake Hillarpintroducesfluorescentmicroscopicmethods to studyceltiitar distribu-tion of biogentc

1956Ritalevi-Montalclnl a

isolate and purifynerve growthfactor.

Single-cell recording. Vernon Mountcastle. David Hubel, andTorsten Wiesel pioneered cellular studies of sensory cortex.

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Big picture. Brain maps,like this one of the ma-caque visual system, revealmultiple brain regionsworking together as a func-tional system.

Drosophlia,and C. eiegans,are introducedto analyzeelementaryaspects of be-havior and learn-ing at thecellular and

lecular

These findings, based on recordings from small neuronalpopulations, have illuminated important issues in perceptionand action. They illustrate how retinal signals are remapped

from retinotopic space into otli-er coordinate frames that canguide behavior; how attentioncan modulate neuronal activity:and how meaning and contextinfluence neuronal aetivity. sothat the same retinal stimuluscan lead to different neuronalresponses depending on howthe stimulus is perceived (28).This same kind of work (relat-ing cellular activity directly toperception and action) is cur-rently being applied to the so-called binding problem—howthe multiple features of a stimu-lus object, which are represent-ed by specialized and distribut-ed neuronal groups, are synthe-sized into a signal that repre-sents a single percept or actionand to the fimdamental questionof what aspects of neuronal ac-tivity (e.g., firing rate or spiketiming) constitute the neuralcodes of infonnation process-ing (29).

Striking parallels to the orga-nization £ind lunction of sensory cortices have been lbund inthe cortical motor areas supporting voluntary movement.Tlius. there are several cortical areas directed to the planningand execution of voluntary movement. Primary motor cortexhas columnar organization, with neurons in each column gov-erning movements of one or a few joints. Motor areas receiveinput from other cortical regions, and information movesthrough stages to the spinal eord, where the detailed eircuitrythat generates motor patterns is located (30).

Although studies of single eells have been enormously in-formative, the functioning brain consists of multiple brainsystems and many neurons operating in concert. To monitoraetivity in large populations of neurons, multielectrode arraysas well as cellular and whole-brain imaging techniques arenow being used. These approaches are being supplementedby studying the effect of selective brain lesions on behaviorand by molecular methods, such as the delivery of markers orother molecules to specific neurons by viral transfection.which promise fine-resolution tracing of anatomical connec-tions, activity-dependent labeling of neurons, and ways totransiently inactivate speeific components of neural circuits.

Invasive molecular manipulations of this kind cannot beapplied to humans. However, functional neuroimaging bypositron emission tomography (PET) or functional magneticresonanee imaging {¡"MRI) provides a way to monitor largeneuronal populations in awake humans while they engage incognitive tasks (31). PET involves measuring regional bloodtlow using H; '•"'0 and allows for repeated measurements onthe same individual. fMRI is based on the fact that neuralactivity changes local oxygen levels in tissue and that oxy-genated and deoxygenated hemoglobin have different mag-netic properties. It is now possible to image the second-by-second time course of the brain's response to single stimulior single events with a spatial resolution in the millimeter

range. Recent sueeess in obtaining fMRI images fromawake monkeys, combined with single-cell recording,should extend the utility of functional neuroimaging by per-mitting parallel studies in humans and nonhuman primates.

One example of how parallel studies of humans ;md non-human primates have advanced the understanding of brainsystems and cognition is in the study of memory. The neuro-seience of memory came into focus in the 1950s when thenoted amnesic patient H.M. was first described (32). H.M.developed profound tbrgetfulness after sustaining a bilateralmedial temporal lobe resection to relieve severe epilepsy. Yethe retained his intelligence, perceptual abilities, and person-alit>. Brenda Milner's elegant studies of H.M. led to severalimportant principles. First, acquiring new memories is a dis-tinct eerebral funetion, separable from other perceptual andcognitive abilities. Second, because H.M. could retain anumber or a visual image for a short time, the medial tem-poral lobes are not needed for immediate memory. Thirdthese structures are not the ultimate repository of memor>.because H.M. retained his remote, childhood memories.

It subsequently became clear that only one kind of mem-ory, declarative memory, is impaired in H.M. and other am-nesic patients. Thus, memory is not a unitary faculty of themind but is composed of multiple systems that have differ-ent logie and neuroanatomy (33). The major distinction isbetween our capacity for conscious, declarative memor\'about facts and events and a collection of unconscious, non-dechiTdtive memory abilities, sueh as skill and habit learningand simple forms of conditioning and sensitizaron. In theseeases, experience modifies performance without requiringany conscious memory content or even the experience thatmemory is being used.

An animal model of human amnesia in the nonhiiman pri-mate was achieved in the early 1980s, leading ultimately tothe identification of the medial temporal lobe structures thatsupport declarative memory—the hippocampus and the adja-eent entorhinal. perirhinal, and parahippocampal cortices(34). The hippocampus has been an especially active target ofstudy, in part because this was one of the structures damagedin patient H.M. and also beeause of the early discovery ofhippocampal place cells, which signal the location of an ani-mal in space (35), This work led to the idea that, onee learn-ing occurs, the hippocampus and other medial temporal lobestructures permit the transition to long-term memory, per-haps by binding the separate cortical regions that togetherstore memory for a whole event. Thus, long-tenn memory isthought to be stored in the same distributed set of corticalstruetiires that perceive, process, and analyze what is to be re-membered, ;md aggregate changes in large assemblies of eor-tical neurons are the substrate of long-term memory. Thefrontal lobes are also thought to influence what is selected forstorage, the ability to hold information in mind for the shortterm, and the ability later on to retrieve it (36).

Whereas declarative memory is tied to a particularbrain system, nondeclarative memory refers to a collectionof learned abilities with ditTerent brain substrates. For ex-ample, many kinds of motor learning depend on the cere-bellum, emotional learning and the modulation of memorystrength by emotion depend on the amygdala, and habitlearning depends on the basal ganglia (37). These forms ofnondeclarative memory, which provide for myriad uncon-scious ways of responding to the world, are evolutionarilyaneient and observable in simple invertebrates such asAply.sia and Dro.sophikt. By virtue of the unconscious sta-tus of these forms of memory, they create some of the

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mystery of human experience. For here arise the disposi-tions, habits, attitudes, and preferences that are inaccessi-ble to conscious recollection, yet are shaped by past events.influence our behavior and our mental life, and are a fun-damental part of who we are.

Bridging Cognitive Neuroscience and Molecular Biology inthe Study of Memory StorageThe removal of scientifie barriers at the two poles of thebiologicLil sciences—in the cell and molecular biology ofnerve cells on the one hand, and in the biology of cognitiveprocesses on the other—has raised the question: Can oneantieipate an even broader unification, one that rangesfrom molecules to mind? A beginning of just such a syn-thesis may be apparent in the study of synaptic plasticityand memory storage.

For all of its diversity, one can view neuroseience as beingconcerned with two great themes—the brain's "hard wiring"and its capacity for plastieity. The fonner refers to how con-nections develop between cells, how cells ftinction and com-municate, and how an organism's inbom ftinctions are orga-nized— its sleep-wake cycles, hunger and thirst, and its abil-ity to perceive the world. Thus, throughevolution the nervous system lias inheritedmany adaptations that are too important tobe Icíí to the vagaries of individual experi-ence. In contrast, the capacity tor plasticityrefers to the fact that nervotLs systems canadapi or change as the result of the experi-ences that occur during an individual life-time. Experience can modify the nervoussystem, and as a result organisms can learnand remember.

The precision of neural connections pos-es deep problems for the plasticity of behav-ior. How does one reconcile the precisionand specificity of the brain's wiring with theknown capability of humans and animals toacquire nev ' knowledge? And how is knowl-edge, once acquired, retained as long-termmemory'.' A key insight about synaptic trans-mission is that the precise eonneetions between neurons are notfixed but are modifiable by experience. Beginning in 1970,studies in invertebrates such as Aplysia showed thai simpleforms of leiiming—liabituation. sensitization, and ckissicaJ con-ditioning—result in functional and structural changes at.synapses between the neurons that mediate the behavior beingmodified. These changes can persist tor days or weeks and par-allel the time courîie of tlic memory process (3H). Tliese cell bi-ological studies have been complemented by genetic sUidies inDfusophila. As a result, studies in .Aplvsia and Dmsophila haveidentified a niunber of proteins importiint for memory (39).

In his now-famous book. The Organization of Behavior,Donald Hebb proposed in 1949 that the synaptic strengthbetween two neurons should increase when the neurons ex-hibit coincident activity (40). In 1973, a long-lasting synap-tic plasticity of this kind was discovered in the hippocampus

I (a key structure for declarative memory) (41). In response toü a burst of high-frequency stimuli, the major synaptic path-^ ways in the hippocampus undergo a long-term change,I known as long-term potentiation or LTP. The advent in theî 1990s of the ability to genetically modify mice made ii pos-« sible to relate specific genes both to synaptic plasticity andt to intact animal behavior, including memory. These tech-S niques now allow one to delete specific genes in specific

brain regions and also to rum genes on and off. Such genetieand pharmacological experiments in intact animals suggestthat interference with LTP at a specific synapse—the Schaf-fer collateral-CAl synapse^—commonly impairs memoryfor space and objects. Conversely, enhancing LTP at thesame synapse can enhance memory in these same declara-tive memory tasks. The findings emerging from these newmethods (42) complement those in Aplysia and Drosophiiaand reinforce one of Cajal's most prescient ideas: Eventhough the anatomical connections between neurons developaccording to a definite plan, their strength £tnd effectivenessare not predetermined and can be altered by experience.

Combined behavioral and molecular genetic studies inDrosophila, Aplysia. and mouse suggest that, despite theirdifferent logic and neuroanatomy. declarative and non-declarative forms of memory share some common cellularand molecular features. In both systems, memory storagedepends on a short-term process lasting minutes and along-term process lasting days or longer. Short-term memo-ry involves covalent modifications of preexisting proteins,leading to the strengthening of preexisting synaptic connec-tions. Long-term memory involves altered gene expression,

protein synthesis, and the growth ofnew synaptic connections. In addition.a number of key signaling moleculesinvolved in converting transient short-term plasticity to persistent long-termmemory appear to be shared by bothdeclarative and iiondeclarative memo-ry. A striking feature of neural plastici-ty is that long-term memory involvesstructural and functional ehange (38.43). This has been shown most directlyin invertebrates and is likely to apply tovertebrates as well, including primates.

It had been widely believed that thesensory and motor cortices mature ear-ly in life and thereafter have a fixed or-ganization and connectivity. However,it is now clear that these cortices can bereshaped by experience (44). In one ex-

periment, monkeys learned to discriminate between two vi-brating stimuli applied to one finger. After several thousandtrials, the cortical representation of the (rained finger be-came more than twice as large as the corresponding areasfor other fingers. Similarly, in a neuroimaging study ofright-handed string musicians the cortical representations ofthe fingers of the left hand (whose fingers are manipulatedindividually and are engaged in skillful playing) were largerthan in nonmusicians. Thus, improved finger skills even in-volve changes in how sensory cortex represents the fingers.Because all organisms experience a different sensory envi-ronment, each brain is modified differently. This gradualereation of unique brain architecture provides a biologicalbasis for individuality.

Coda

Physicists and chemists have often distinguished their disci-plines from the field of biology, emphasizing that biologywas overly descriptive, atheoreticai, and lacked the coher-ence of the physical sciences. This is no longer quite true.In the 20th eentury. biology matured and became a coherentdiscipline as a result of the substantial achievements ofmolecular biology. In the second half of the eentury. neuro-seience emerged as a discipline that concerns itself with

Real-time brain. Imaging methods canreveal those brain areas that are activeduring specific cognitive tasks.

1962-63Brain anatomy inrodents is foundto be altered byexperience; firstevidence for roteof protein syn-thesis in meaiformation.

1963Roger Sperry pro-poses a precisesystem of chemi-cat matching be-tween pre- andpostsynapticneuronal partners(the chemoaffini-ty tiypothests).

1966-69Ed Evarts andRobert Wurt i de-velop methodstor studyingmovement anaperception withsingle-cetlrecordings fromäwake, behavingmonkeys.

1970Synaptic chaiare related toLearning andmemory storagein Apiyiia.

Mid-1970sPaul Creengardshows that manyneuro transmit-ters work bymeans of proteinphosphorylation.

1973Timothy Bliss andTerje Lomo dis-cover long termpotentiation, aCandidate synap-tic mechanism fortong-term mam-maiian memory,

1976 ^Erwin Neher aSert Sakmann de-velop the patch-clamp techniquefor recording theactivity of 5ingleion channels.

Late 19701Neuroimaging bypositron emissiontomography Isdeveloped,

1980sExpérimentât evi-dence becomesavailable for thedivisibility ofmemory intomultiple sys t«—an animal mo'of human amisia is developed.

vww.sciencemag.org SCIENCE VOL 290 10 NOVEMBER 2000 1119

P A T H W A Y S O F D I S C O V E R Y

1986H.Robert Horvitzdiscovers the cedgenes, which ar«critical for pro-grammed cetl

ÏPatient R.B.«-tabtishes the im-portance of thehippocampus for

' human memory.

1990it$6gj Ogawa ano; colleagues deveUI op functionalt magnetic reso-J, nance Imaging.

, Mario Capetchiand Oliver

• Smythtes develop^ gene knockout\\ technotogy, whichI Is soon applied toI ncuroscwnce.

1991lind« Buck and

' Richard Axel dis-' cover that the ol-

factory receptorfamily consists ofover TOOO differ-ent gerws. Theanatomical com-ponents of themedial temporallobe memory sys-tem «re identified.

1993The Huntington'sDisease Collabo-

!• rative Research, Croup identifies

the gene respon-. siblefor Hunting-

1990sNeural develop-

- ment is trans-formed from a de-scriptive to amolecular disci-pline by GeraldFischbach, jackMcMahan.TomJessell. and CoreyGoodman; neu-roimaging is ap-plied to problemsof human cogni-tion, inciudirtgperception, atten-tion, and memory

Reinhard Jahn,James Rothman,Richard Schelter,and Thomas Sud-hof delineatemolecules critical' exocytosis

Fir« 3D structureof an Ion channel

¡< is revealed byRod MacKinnon.

both biology and psychology and that isbeginning to achieve a similar coher-ence. As a result, fascinating insightsinto the biology of cells, and remark-able principles of evolutionary conser-vation, are emerging from the study ofnerve cells. Similarly, entirely new in-sights into the nature of mental process-es (perception, memory, and cognition) are emerging fromthe study of neurons, circuits, and brain systems, and com-putational studies are providing models that can guide ex-perimental work. Despite this remarkable progress, the neu-roscience of higher cognitive processes is only beginning.For neuroscience to address the most challenging problemsconlronting the behavioral and biological sciences, we willneed to continue to search for new molecular and cellularapproaches and use them in conjunction with systems neu-roscience and psychological science. In this way, we willbest be able to relate molecular events and spécifie changeswithin neuronal circuits to mental processes such as percep-tion, memory, thought, and possibly consciousness itself.

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45. The work of E.R,K. is supported by NIMH, the C. Harold and Leila Y. Math-ers Foundation, the Lieber Center for Research on Schizophrenia, and theHoward Hughes Medical Institute. The work of L.R.S. is supported by theMedical Research Service of the Department of Veterans Affairs, NIMH,and the Metropolitan Life Foundation. We thank Thomas Jessell andThomas Albright for their helpful comments on the manuscript.

Eric R. Kandel is University Professor, Columbia University, New York, andSenior Investigator at the Howard Hughes Medical Institute. He is a mem-ber of the National Academy of Sciences and the Institute of Medicine anda past president of the Society for Neuroscience. This past October, he wasnamed a co-recipient of the Nobel Prize in physiology or medicine,

Larry R. Squire is Research Career Scientist at the Veterans Affairs SanDiego Healthcare System and Professor of Psychiatry, Neurosciences, andPsychology, University of California, San Diego. He is a member of theNational Academy of Sciences and the Institute of Medicine and a pastpresident of the Society for Neuroscience,

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