mechanis of the memory

NEUROBIOLOGY OF LEARNING AND MEMORY 68, 285–316 (1997)ARTICLE NO. NL973799

Memory Formation: The Sequence of BiochemicalEvents in the Hippocampus and Its Connection

to Activity in Other Brain Structures

Ivan Izquierdo* and Jorge H. Medina†,1

*Departamento de BioquıB mica, Instituto de Ciencias Basicas da Saude, Universidade Federaldo Rio Grande do Sul, Ramiro Barcellos 2600, 90035-003 Porto Alegre, RS, Brazil; and†Laboratorio de Neurorreceptores, Instituto de BiologıB a Celular, Facultad de Medicina,

Universidad de Buenos Aires, Paraguay 2155, 3er Piso, Buenos Aires, Argentina

Recent data have demonstrated a biochemical sequence of events in the rat hippocam-pus that is necessary for memory formation of inhibitory avoidance behavior. Thesequence initially involves the activation of three different types of glutamate receptorsfollowed by changes in second messengers and biochemical cascades led by enhancedactivity of protein kinases A, C, and G and calcium–calmodulin protein kinase II,followed by changes in glutamate receptor subunits and binding properties and in-creased expression of constitutive and inducible transcription factors. The biochemicalevents are regulated early after training by hormonal and neurohumoral mechanismsrelated to alertness, anxiety, and stress, and 3–6 h after training by pathways relatedto mood and affect. The early modulation is mediated locally by GABAergic, cholinergic,and noradrenergic synapses and by putative retrograde synaptic messengers, and ex-trinsically by the amygdala and possibly the medial septum, which handle emotionalcomponents of memories and are direct or indirect sites of action for several hormonesand neurotransmitters. The late modulation relies on dopamine D1, b-noradrenergic,and 5HT1A receptors in the hippocampus and dopaminergic, noradrenergic, and seroto-ninergic pathways. Evidence indicates that hippocampal activity mediated by gluta-mate AMPA receptors must persist during at least 3 h after training in order formemories to be consolidated. Probably, this activity is transmitted to other areas,including the source of the dopaminergic, noradrenergic, and serotoninergic pathways,and the entorhinal and posterior parietal cortex. The entorhinal and posterior parietalcortex participate in memory consolidation minutes after the hippocampal chain ofevents starts, in both cases through glutamate NMDA receptor-mediated processes,and their intervention is necessary in order to complete memory consolidation. Thehippocampus, amygdala, entorhinal cortex, and parietal cortex are involved in retrievalin the first few days after training; at 30 days from training only the entorhinal andparietal cortex are involved, and at 60 days only the parietal cortex is necessary forretrieval. Based on observations on other forms of hippocampal plasticity and on mem-ory formation in the chick brain, it is suggested that the hippocampal chain of eventsthat underlies memory formation is linked to long-term storage elsewhere throughactivity-dependent changes in cell connectivity. q 1997 Academic Press

1 This work was supported by PRONEX, Brazil, and University of Buenos Aires, Argentina.Address correspondence and reprint requests to Dr. Ivan Izquierdo, Departamento de Bio-quıB mica, Instituto de Ciencias Basicas da Saude, Universidade Federal do Rio Grande do Sul, Av.Ramiro Barcellos 2600, 90035-003 Porto Alegre, RS, Brazil. Fax: 55 51 316 5535.

285 1074-7427/97 $25.00Copyright q 1997 by Academic Press

All rights of reproduction in any form reserved.

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286 IZQUIERDO AND MEDINA

Step-down inhibitory (passive) avoidance learning in the rat triggers bio-chemical events in the hippocampus that are necessary for the retention ofthis task. The events are similar in many ways to those described for differenttypes of long-term potentiation (LTP) and other forms of neural plasticity(Baudry, Bi, & Tocco, 1996; Bliss & Collingridge, 1993; Collingridge & Bliss,1993; Izquierdo & Medina, 1995, 1997; Maren & Baudry, 1995; Martin &Kandel, 1996; Reymann, 1993). They are triggered by glutamate receptor acti-vation and involve at least four different biochemical cascades led by differentprotein kinases: Protein kinase G (PKG) (Bernabeu, Schroder, Quevedo, Cam-marota, Izquierdo, & Medina, 1997c), protein kinase C (PKC) (Bernabeu, Iz-quierdo, Cammarota, Jerusalinsky, & Medina, 1995a; Bernabeu, Cammarota,Izquierdo, & Medina, 1997b), calcium–calmodulin-dependent protein kinaseII (CaMKII) (Bernabeu et al., 1997b; Wolfman, Fin, Dias, Bianchin, Da Silva,Schmitz, Medina, & Izquierdo, 1994), and protein kinase A (PKA) (Bernabeu,Bevilaqua, Ardenghi, Bromberg, Schmitz, Bianchin, Izquierdo, & Medina,1997a; Bevilaqua, Ardenghi, Schroder, Bromberg, Schmitz, Schaeffer, Que-vedo, Bianchin, Walz, Medina, & Izquierdo, 1997) (Fig. 1).

Several steps of these cascades have been implicated in other forms of learn-ing that also involve the hippocampus (Abel, Nguyen, Barad, Deuel, Kandel &Bourchuladze, 1997; Bliss & Collingridge, 1993; Guzowski & McGaugh, 1997;Izquierdo, Da Cunha, Rosat, Jerusalinsky, Ferreira, & Medina, 1992; Iz-quierdo & Medina, 1995; Mayford, Bach, Huang, Wang, Hawkins, & Kandel,1996; Nogues, Micheau, & Jaffard, 1994; Tan & Liang, 1996; Tocco, Devgan,Hauge, Weiss, Baudry, & Thompson, 1991; Wilson & Tonegawa, 1997), inanother form of inhibitory avoidance in the chick brain (Anokhin, Mileusnic,Shamakina, & Rose, 1991; Rose, 1995a,b; Zhao, Polya, Wang, Gibbs, Sed-man, & Ng, 1995), and in other forms of neural plasticity (Abel et al., 1997;Bartsch, Ghirardi, Skehel, Karl, Herder, Chen, Bailey, & Kandel, 1995; Be-ninger & Nakonechny, 1996; Bliss & Collingridge, 1993; Carew, 1996; Huang,Colley, & Routtenberg, 1992; Reymann, 1993; Yin & Tully, 1996). Most of theevidence for the role of these processes in memory formation comes from stud-ies on the effect on memory of infusions of specific receptor agonists and antago-nists or enzyme inhibitors at various times after inhibitory avoidance traininginto the hippocampus and elsewhere and from detailed biochemical or histo-chemical analysis of these receptors and enzymes in the same structures, alsoat various times after training (Izquierdo & Medina, 1995, 1997). Several ofthe findings have been corroborated in other tasks using the same techniques(Izquierdo & Medina, 1995, 1997), and, more recently, by second-generationgene knockout studies in mouse hippocampus (Mayford et al., 1996; Wilson &Tonegawa, 1997).

Step-down inhibitory avoidance involves learning not to step down from aplatform in order to avoid a mild footshock. It is usually acquired in onesingle trial, which makes it ideal for studying processes initiated by training,uncontaminated by prior or further trials, rehearsals, or retrievals (Gold, 1986;Izquierdo, 1989). Step-down avoidance involves the specific repression of thenatural tendency of rats to explore beyond the platform, without affecting theperformance of exploratory behavior while on the platform, repeated approxi-mations to its border, or abortive step-down responses; for these reasons weprefer the term ‘‘inhibitory’’ to ‘‘passive’’ (Netto & Izquierdo, 1985). There aremany variants of inhibitory avoidance: In rodents, not to step through a doorinto a compartment where they receive footshocks; in flies, not to enter a foul-


287HIPPOCAMPAL BIOCHEMISTRY AND MEMORY

odored area; in chicks, not to peck a bitter bead; in humans, to refrain fromputting the fingers in the electrical outlet or from crossing a street withoutlooking. Obviously, this task represents one of the major determinants of sur-vival behavior in all species (Gold, 1986), and the brevity of its acquisitionshould not mislead into thinking that it is inborn or implicit. Indeed, there isa seldom-measured implicit component of this task (bradycardia) that, unlikeits explicit component (increased avoidance latency), is insensitive to electro-convulsive shock (Hine & Paolino, 1970). The declarative component of thistask, like that of many others (Calderazzo Filho, Cavalheiro, & Izquierdo, 1977;Eichenbaum, 1996; Gabrielli, Brewer, Desmond, & Glover, 1997; Izquierdo etal., 1992; Izquierdo & Medina, 1991, 1995; Matthies, 1982, 1989; Squire, 1992;Tocco et al., 1991; Vnek & Rothblat, 1996), crucially involves the hippocampus(Izquierdo et al., 1992; Izquierdo & Medina, 1991, 1995, 1997; Lorenzini, Baldi,Bucherelli, Sacchetti, & Tassoni, 1996; O’Connell, O’Malley, & Regan, 1997).

Carew (1996) recently observed that ‘‘typically, the encoding of a lastingmemory entails considerable practice and rehearsal, but . . .rare events inour life . . .are so powerful as to be instantly recorded and remembered fora lifetime.’’ An afferent volley such as that used in order to induce LTP inrats, or the association of a given experience with a footshock or a strongtaste, such as that used for inhibitory avoidance conditioning in rats andchicks, respectively, or a strong odor such as that used for training in thefly, or a jet of 5HT in the mollusk ganglion certainly constitute ‘‘rare’’ and‘‘powerful’’ events and induce pronounced biochemical changes. It is pre-sumed that similar changes occur to a lesser degree, or in a smaller numberof synapses, in less dramatic memories (Carew, 1996; Izquierdo & Medina,1997; Yin & Tully, 1996). The presumption is based on a large body of evi-dence showing that the effects of many of the drugs that alter the memoryof inhibitory avoidance and many of the hippocampal biochemical changesare also seen following exploratory habituation and other tasks (Izquierdo etal., 1992; Izquierdo & Medina, 1995; Matthies, 1982, 1989; Rose, 1995a;Tan & Liang, 1996; Wolfman, Da Cunha, Jerusalinsky, Levi de Stein, Viola,Izquierdo, & Medina, 1991).

THE HIPPOCAMPAL BIOCHEMICAL CASCADESOF INHIBITORY AVOIDANCE LEARNING

The Initial Role of Glutamate Receptors

The biochemical events underlying memory formation of the inhibitoryavoidance task in the hippocampus involve, first, an activation of NMDA,AMPA, and metabotropic glutamate receptors, and a short-lasting increaseof NMDA1 levels and a longer lasting increase of GluR1 levels (see below)(Izquierdo & Medina, 1995, 1997; Medina & Izquierdo, 1995). Immediate butnot delayed (30–180 min) intrahippocampal infusion of the NMDA antagonistaminophosphonopentanoic acid (AP5) (Izquierdo et al., 1992; Jerusalinsky,Ferreira, Da Silva, Bianchin, Ruschel, Medina, & Izquierdo, 1992) or of theglutamate metabotropic receptor antagonist methyl-carboxyphenyl glycine (Bi-anchin, Da Silva, Schmitz, Medina, & Izquierdo, 1994) is amnestic. Immediateposttraining intrahippocampal infusion of glutamate or of the metabotropicagonist aminocyclopentane dicarboxylate causes retrograde memory facilita-tion (Bianchin et al., 1994; Izquierdo et al., 1992). Intrahippocampal adminis-



tration of the AMPA receptor antagonist cianonitroquinoxaline-dione (CNQX)causes amnesia for the inhibitory avoidance task when given up to 3 h aftertraining (Jerusalinsky et al., 1992), which indicates that hippocampal excita-tion mediated by these receptors is necessary for memory formation duringthat period.

The role of hippocampal NMDA receptors in several forms of learning, partic-ularly those that involve distant visual cues, has been studied by severalauthors (see references in Bliss & Collingridge, 1993; Izquierdo & Medina,1995, 1997; Reymann, 1993). Actually, the first authors to suggest this wereMorris, Anderson, Lynch, and Baudry (1986), studying the effect of chronicintracerebroventricular AP5 release by a micropump on spatial learning in awater maze. Recently, selective knockout of the gene that encodes for theNMDA1 receptor subunit in mouse CA1 was shown to disrupt both place cellensemble activity and memory of spatial memory (Wilson & Tonegawa, 1997).A similar previous finding had been obtained using first-generation (wholebody) deletion of the NMDAR1 gene (Sakimura, Kutsuwada, Ito, Manabe,Takayama, Kushiya, Yagi, Alzawa, Inoue, Sugiyama, & Mishina, 1995). Therole of hippocampal NMDA receptors in memory formation has been discussedby numerous authors (see Izquierdo & Medina, 1995, 1997, for references).The role of hippocampal AMPA receptors in memory formation was discussedby Jerusalinsky et al. (1992) and by Cammarota, Bernabeu, Izquierdo, andMedina (1996). The role of metabotropic glutamate receptors in memory forma-tion in this and other tasks has been recently discussed by Reymann (1993)and Riedel (1996).

Early Involvement of Putative Retrograde Messengers

Evidence suggests a participation of the putative retrograde messengersthat regulate glutamate release, nitric oxide (NO), carbon monoxide (CO), andthe platelet activating factor (PAF) in the early phase of memory formation ofthe step-down task in the hippocampus (Medina & Izquierdo, 1995). The roleis much clearer than the one proposed for these substances in LTP induction(see Bliss & Collingridge, 1993; Medina & Izquierdo, 1995; Son, Hawkins,Martin, Kiebler, Huang, Fishman, & Kandel, 1997). The immediate but notthe delayed (30 min) intrahippocampal infusion of the NO releaser S-nitroso-N-aminopenicillin (Bernabeu, Levi de Stein, Fin, Izquierdo, & Medina, 1995b)or of a soluble form of the platelet activating factor (mcPAF) (Izquierdo, Fin,Da Silva, Jerusalinsky, Quillfeldt, Ferreira, Medina, & Bazan, 1995) causesmemory facilitation. The activity of NO synthase (Bernabeu et al., 1995b), andof heme oxygenase, the enzyme that produces CO (Bernabeu, Levi de Stein,Princ, Fin, Juknat, Batlle, Izquierdo, & Medina, 1995c), sharply increasesimmediately after training and returns to normal a few minutes later. Immedi-ate but not delayed (30 min) infusion into CA1 of a PAF antagonist (Izquierdoet al., 1995), of a NO synthase inhibitor (Bernabeu et al., 1995b), or of a hemeoxygenase inhibitor (Bernabeu et al., 1995c) causes retrograde amnesia forthis task.

Early Modulation by GABAA and Other Receptors

In the first few minutes after training, memory formation is very sensitiveto inhibition by GABAA receptors (when given into CA1, muscimol is amnesticand picrotoxin facilitates memory) and to modulation by cholinergic muscarinic



and b-noradrenergic receptors (when given into CA1, norepinephrine and oxo-tremorine facilitate memory, scopolamine is amnestic, and timolol alters sensi-tivity to GABAergic modulation; Izquierdo et al., 1992).

Changes in Glutamate Receptor Properties

In synaptic membranes extracted from CA1 there is a short-lived increaseof NMDA1 levels measured by immunoblot at 30 min from training, and aslow rise of the levels of GluR1 that starts about 30 min after training andpeaks at approximately 3 h (Bernabeu et al., 1997c). NMDA1 is a specificsubunit of NMDA receptors, and GluR1 is a specific subunit of AMPA receptors.The GluR1 increase occurs concomitantly with a slow rise of Bmax of AMPAto AMPA receptors in synaptic membranes extracted from all hippocampalsubregions (Cammarota, Izquierdo, Wolfman, Levi de Stein, Bernabeu, Jeru-salinsky, & Medina, 1995) that lasts about 3 h in CA1 and CA2, but as muchas 168 h in CA3 and in the dentate gyrus (Cammarota et al., 1996). IncreasedAMPA binding has been reported in the hippocampus after LTP by Tocco,Maren, Shors, Baudry, and Thompson (1992) and by Tocco et al. (1991) follow-ing eye-blink conditioning. Sergueeva, Fedorov, and Reymann (1993) havereported increased electrophysiological sensitivity of hippocampal AMPA re-ceptors to AMPA in the first 90 min after LTP. The significance of such changesis not known, but no doubt they reflect increased excitability of hippocampalneurons, which may underlie plastic changes in their output to projection sites.

The increase of GluR1 levels measured by immunoblot in CA1 synaptosomalmembranes seen after training may correlate with phosphorylation of the sub-unit by CaMKII and PKA (see below). As will be commented further on, theactivity of these two enzymes in hippocampus increases right after training.Incubation of synaptic membranes under conditions of optimum phosphoryla-tion by CaMKII and/or PKA increases not only phosphorylation of the subunit,but also its Bmax for AMPA and the actual amount of GluR1 measurable byimmunoblot (Bernabeu et al., 1997b). The regulation of AMPA (and NMDA)receptors by PKA- and CaMKII-mediated phosphorylation has been well estab-lished in various synapses of the rat brain (McGlade-McCulloh, Yamamoto,Tan, Brickey, & Soderling, 1993; Pasqualotto & Shaw, 1996; Roche, O’Brien,Mammen, Bernhardt & Huganir, 1996).

Involvement of Extrahippocampal Structures

Several of the findings mentioned above (effect of AP5, CNQX, picrotoxin,muscimol, oxotremorine, scopolamine, norepinephrine, timolol, and PAF) havebeen also observed studying immediate posttraining infusions into the amyg-dala and medial septum (see Izquierdo et al., 1992; Izquierdo & Medina, 1995;Walz, Da Silva, Bueno e Silva, Medina, & Izquierdo, 1992). This led to thesupposition that LTP-like phenomena in these other structures might be in-volved in memory consolidation, alongside those in the hippocampus (Izquierdoet al., 1992; Jerusalinsky et al., 1992), a supposition that later evidence provederroneous (see below and Izquierdo & Medina, 1997). Ample evidence suggeststhat the amygdala (Bevilaqua et al., 1997; Cahill & McGaugh, 1990, 1996;Gray, 1982; Izquierdo & Medina, 1991, 1997; Walz et al., 1992) and to anextent the medial septum (Gray, 1982; Izquierdo & Medina, 1991, 1997; Walzet al., 1992; Wolfman et al., 1991) are involved in the processing of anxiety,alertness, or aversiveness.



FIG. 1. This and all the following figures show biochemical data of hippocampal CA1 regionsof rats sacrificed at various times after step-down inhibitory avoidance training with a 0.3-mAfootshock, and test session performance data of rats that received bilateral drug infusions intoCA1 at various times after training. Time of sacrifice or infusion is shown in the abscissae, inmin. The biochemical data in the ordinates are expressed as means { SEM % of shocked controls(animals placed on the grid of the apparatus and exposed just to the footshock without avoidancetraining). The behavioral data are means { SEM test minus training step-down latencies ex-pressed as a % of those from saline- or vehicle-treated controls. Asterisks indicate significantdifferences from controls in Newman–Keuls tests: *p õ .01 and **p õ .05. This figure showsdata relevant to the cGMP/PKG cascade: cGMP levels measured by radioimmunoassay in CA1(tilted solid squares), PKG activity in CA1 measured using peptide G-5611 as substrate (solidsquares), and behavioral effects of LY83583 (2.5 mg/side) or 8-Br-cGMP (1.25 mg/side) infusedinto CA1. Both cGMP levels and PKG activity rise immediately after training and return tonormal 30 min later. LY83583 causes retrograde amnesia, and 8-Br-cGMP causes retrogradememory facilitation, when given 0 but not 30 min posttraining. Data are from Bernabeu et al.(1996, 1997c). These findings point to an early, rapid, and necessary intervention of cGMP andPKG in memory consolidation.

The cGMP/PKG Cascade

NO and CO activate soluble guanylyl cyclase, the enzyme that synthesizescyclic guanosyl monophospate (cGMP) (Zhuo, Hu, Schultz, Kandel, & Hawkins,1994a). NO, CO, and cGMP may induce long-lasting synaptic potentiation inhippocampal CA1 (Zhuo, Kandel, & Hawkins, 1994b) and the cGMP proteinkinase (PKG) appears to be crucial for CA1 LTP (Zhuo et al., 1994a).

The immediate but not the delayed (30–180 min) infusion into CA1 of 8-Br-GMP causes memory facilitation of step-down avoidance (Bernabeu, Schmitz,Faillace, Izquierdo, & Medina, 1996), whereas that of the guanylyl cyclaseinhibitor LY83583 is amnestic (Bernabeu et al., 1997c). Guanylyl cyclase activ-ity increases immediately after training and returns to normal 30 min later(Bernabeu et al., 1997c) (Fig. 1). Endogenous cGMP levels in rat CA1 increasemarkedly, but very briefly, soon after inhibitory avoidance training and returnto normal levels between 30 and 60 min later (Bernabeu et al., 1996). Thus,



perhaps as in LTP (Zhuo et al., 1994a), the evidence indicates that the cGMP/PKG cascade in CA1 is crucial early on for the long-term plasticity of inhibitoryavoidance (Kim, 1996).

There is so far no indication of what PKG substrates may be involved inmemory processing in the first few minutes that follow acquisition. In LTP,presynaptic guanylyl cyclase is supposedly a target of NO and CO action,and PKG activation might help in the mobilization of glutamatergic synapticvesicles toward the synaptic cleft (see Zhuo et al., 1994a, and Medina & Iz-quierdo, 1995, for references). These possibilities await further study.

The CaMKII Cascade

CaMKII activity is regulated by the increase of intracellular Ca2/ thatfollows activation of NMDA receptors or the entry of Ca2/ through voltage-dependent channels that trigger LTP in CA1 and in CA3, respectively, or inother areas of the brain (Bliss & Collingridge, 1993). Not surprisingly,CaMKII has been suggested early on to play a key role in the early postinduc-tion phase of LTP (Reymann et al., 1988), which was confirmed by laterstudies using specific inhibitors (Ito, Hidaka, & Sugiyama, 1991) and enzymeassays (Fukunaga, Stoppini, Miyamoto, & Muller, 1993), as well as first-generation (whole body; Gordon, Cioffi, Silva, & Stryker, 1996) and second-generation transgenic studies (localized to CA3, Mayford et al., 1996, respec-tively). The time course of the intervention of CaMKII in hippocampal CA1or CA3 LTP more or less overlaps with that of PKC, except that the peak ofCaMKII activity occurs earlier and the increase of activity lasts less thanthat of PKC (see Reymann, 1993).

CaMKII mediates the phosphorylation of a variety of proteins of importancein synaptic plasticity, including the ionotropic glutamate receptors (see above),CREB (Ferrer, Blanco, Rivera, Carmona, Ballabriga, Olive, & Planas, 1996),and neurofilaments and other structural proteins (Schroder, de Mattos-Dutra,de Freitas, Lisboa, Zilles, Pessoa-Pureur, & Izquierdo, 1997). In all these cases,it acts concomitantly with PKA; however, since the sites of each of these pro-teins that are phosphorylated by the two enzymes are different, the functionssubserved by CaMKII and PKA are also different. For reviews of these aspects,see Ferrer et al. (1996), Pasqualotto and Shaw (1996), Roche et al. (1996), andSchroder et al. (1997).

The first evidence for an involvement of CaMKII in memory processing inhippocampus and amygdala was provided by Wolfman et al., (1994), whoinfused the specific inhibitor of this enzyme, KN62, into CA1 or into theamygdala at different times after step-down inhibitory avoidance training.A very strong amnestic effect was obtained when the inhibitor was infusedimmediately posttraining into hippocampus or amygdala. Intrahippocampalinfusion of the drug 30 min after training had only a partial amnestic effect,and infusions 2–4 h after training into either structure were ineffective. Thissuggested that CaMKII plays an essential role in the hippocampus (and amyg-dala) in the first few minutes after training, as it does in LTP in the firstfew minutes after induction (Ito et al., 1991; Reymann, Brodemann, Kase, &Matthies, 1988).

Subsequently, Tan and Liang (1996) reported similar findings on the amnes-tic effect of CaMKII inhibitors in a spatial learning task and showed a post-training increase of CaMKII activity following training in that task. We were



FIG. 2. The CaMKII cascade. Ca2/-dependent (shaded squares) and Ca2/-independent(crosses) CaMKII activity in CA1 measured using Syntide-2 as substrate, CA1 levels of the AMPAreceptor subunit, GluR1 measured by immunoblot (open squares) and GluR1 phosphorylation(tilted crosses), [3H]AMPA binding to AMPA receptors in CA1 (bars), and behavioral effects ofCNQX (1.25 mg/side, solid triangles) and of the CaMKII inhibitor KN62 (3.6 mg/side; open triangles)infused into CA1. CaMKII activity increased in the first half hour after training; the Ca2/-depen-dent activity increased more and remained high for a longer time than the Ca2/-independentactivity. GluR1 levels, GluR1 phosphorylation, and AMPA binding increased gradually from 30min after training on. Both CNQX and KN62 caused retrograde amnesia; the former, when givenup to 180 min posttraining, the latter only when given up to 180 min posttraining, the latter onlywhen given immediately after training, and only partially when given 30 min later. Data fromJerusalinsky et al. (1992), Wolfman et al. (1994), Cammarota et al. (1996, 1997), and Bernabeuet al. (1997b). The data show a necessary participation of CaMKII in memory consolidation inthe first few min after training and of AMPA receptors from 0 to 3 h after training. Various datain the literature suggest that the latter depends on the former (see Bernabeu et al., 1997b).

able to confirm and complement these findings using the step-down inhibitoryavoidance task (Fig. 2) (see Bernabeu et al., 1997b). Ca2/-dependent a-CaMKIIactivity increased markedly right after training, decreased slightly but stillremained high at 30 min, and returned to normal in 120 min (Bernabeu etal., 1997b). These data are the mirror image of those that were obtained byWolfman et al. (1994) using Kn62. In addition, we found that Ca2/-independentb-CaMKII activity increased slightly immediately after training and returnedto normal in 30 min. Autophosphorylation of the enzyme (i.e., conversion fromthe active, Ca2/-dependent to the inactive, Ca2/-independent form) was maxi-mal immediately after training (Bernabeu et al., 1997b).

Changes in the phosphorylation levels of neurofilament proteins of rat hippo-campus, which is mediated by CaMKII and by PKA, have been observed 60min after either step-down inhibitory avoidance training or the habituation ofexploration of the training apparatus (Schroder et al., 1997).



An elegant general confirmation of the importance of a-CaMKII in learningprocesses has been obtained using regulated expression of the CaMKII transgenelocalized to the CA3 region of the hippocampus (Mayford et al., 1996).

As is known, retrograde amnesia for the inhibitory avoidance task can beinduced by a variety of agents, including some that are clearly modulatory,like systemically administered b-endorphin (see Izquierdo, 1989; McGaugh,1989). Recently (L. A. Izquierdo, Schroder, Ardenghi, Quevedo, Bevilaqua,Netto, I. Izquierdo, & Medina, 1997b), we have observed that the retrogradeamnesia caused by systemic b-endorphin administration or by electroconvul-sive shock can be reversed by pretest administrations of adrenocorticotropinor vasopressin (see Bohus, 1994), whereas the amnesia induced by the CaMKIIinhibitor KN62 given intrahippocampally 0 h after training or that inducedby the PKA inhibitor KT5720 given intrahippocampally 3 h after trainingcannot be reversed by the hormones. This further illustrates that CaMKIIand PKA (see next section) participate in the core mechanism, and not in amodulatory mechanism, of memory in the hippocampus.

The PKC Cascade

PKC is activated by various transmitters, including glutamate and acetyl-choline (Bliss & Collingridge, 1993; Jerusalinsky, Kornisiuk, & Izquierdo,1997), and is essential for the continuation of LTP beyond the induction phase(Ben-Ari, Anikstejn, & Bergestovski, 1992; Colley & Routtenberg, 1993; Huanget al., 1992). In LTP, first the postsynaptic g isoform of PKC is activated andthen the presynaptic b isoform, which phosphorylates the presynaptic proteinGAP-43 (Routtenberg, Lovinger & Stewart, 1985). PKC inhibitors given earlyafter induction block LTP (Huang et al., 1992; Reymann et al., 1988). Oneimportant feature of PKC is that, like Ca2/, it stimulates adenylyl cyclase andtherefore cAMP synthesis (Yoshimura & Cooper, 1993), which, as will be seenbelow, plays a crucial role in the activation of PKA and the triggering ofanother cascade of great importance in the long-term persistence of memoryand other plastic events.

In view of the many similarities between the hippocampal biochemistry ofmemory and that of LTP (Bliss & Collingridge, 1993; Izquierdo & Medina,1995, 1997; Maren & Baudry, 1995; Reymann, 1993), the suggestion arosethat PKC may be involved in memory formation. PKC is redistributed fromcytoplasm to membrane in rat hippocampal CA3 after discrimination learning(Olds, Golski, McPhie, Olton, Mishkin, & Alkon, 1990) and eye-blink condition-ing (Scharenberg, Olds, Schereurs, Craig & Alkon, 1991). Sheu, McCabe, Horn,and Routtenberg (1993) reported increased PKC substrate phosphorylation inspecific regions of the chick brain after inhibitory avoidance. Serrano, Benis-tan, Oxonian, RodrıB guez, Rosenzweig, and Bennett (1994) reported on theamnestic effect of PKC and other protein kinase inhibitors administered intothe chick brain in this task. Actually, abundant proof that memory formationin the chick depends on membrane-bound protein kinase C was provided sev-eral years earlier by Bourchuladze, Potter, and Rose (1990). Nogues et al.(1994) detected increased PKC activity in mouse hippocampus following aspatial task. Transgenic mice with a knockout of the gene that encodes for g-PKC in the whole organism (i.e., first-generation knockouts; see Wilson &Tonegawa, 1997) showed impaired hippocampal LTP (Abeliovich, Chen, Goda,Silva, Stevens & Tonegawa, 1993a) and spatial and contextual learning (Abeli-



FIG. 3. The PKC cascade. PKC activity measured in CA1 by in vitro phosphorylation usinghistone IIIS as substrate (squares), phosphorylation of the PKC substrate GAP-43 in CA1 (trian-gles), and behavioral effect of the PKC inhibitor CGP41231 (2.5 mg/side) infused into CA1. PKClevels rise immediately after training, reach a peak at 30 min, and return to normal at 120 min.GAP-43 phosphorylation reaches a peak at 30 min. CGP41231 is fully amnestic when given 0 or30 min posttraining into CA1, and partially amnestic when given at 120 min. A similar effect hasbeen reported for another PKC inhibitor, staurosporin (Jerusalinsky et al., 1994b). Data fromCammarota et al. (1997) and Jerusalinsky et al. (1994b). The data point to a necessary role ofPKC activity in CA1 in memory consolidation in the first 30 min after training.

ovich, Paylor, Chen, Kim, Wehner, & Tonegawa, 1993b), two declarative tasksthat, like most others, depend on the hippocampus (Eichenbaum, 1996; Eichen-baum, Schoenbaum, Young, & Bunsey, 1996). Criticisms of first-generationtransgenic experiments (developmental variables, adaptive changes, alter-ations all over the body) can be found in Maren and Baudry (1995), Routtenberg(1995), and Wilson and Tonegawa (1997); the LTP and learning changes seenin g-PKC mutants, however, correlate well with those predicted from pharma-cological or biochemical experiments (see below).

We observed that the intrahippocampal (Jerusalinsky, Quillfeldt, Walz, DaSilva, Medina & Izquierdo, 1994b), intra-amygdala, or intraentorhinal (Jeru-salinsky et al., 1992) infusion of two different PKC inhibitors, staurosporinand CGP41231, within the first 120 min after step-down inhibitory avoidancetraining, blocked memory expression of this task measured 24 h later (Fig. 3).The amnesia was complete when the drugs were given into hippocampus oramygdala up to 60 min after training, and partial when the infusion wasdelayed for 120 min. Subsequently, we measured posttraining PKC activityby two different methods: One less specific (phorbol dibutyrate binding; Berna-beu et al., 1995a), and another one very specific (using a synthetic peptidesubstrate; Cammarota, Paratcha, Levi de Stein, Bernabeu, Izquierdo, & Me-dina, 1997; Fig. 3). Both experiments revealed a very large increase of mem-brane-bound PKC activity in hippocampus relative to shocked and naive con-trols, starting immediately after step-down avoidance training, reaching apeak 0.5 h later, and returning to normal values over the next 2.0 h. A similar



pattern of changes was observed, with less intensity, in the amygdala and inthe frontal, parietal, and entorhinal cortex, and to a lesser degree in cerebellum(Bernabeu et al., 1995a). No changes were observed in other brain structures.The coincidence of the time course of the change of hippocampal PKC activitywith that of the amnestic effects of the PKC inhibitors (see above) points toa clear and crucial involvement of this enzyme in the posttraining memoryprocessing of this task.

The function of hippocampal PKC in memory is as unclear as it is in LTP(see Colley & Routtenberg, 1993; Reymann, 1993). As found by Routtenberget al. (1985) following LTP, we detected 30 min after avoidance training a briefpeak of enhanced PKC-mediated phosphorylation of the presynaptic protein,GAP-43, which coincided with the PKC activity peak and was blocked, likethe latter, by diverse PKC inhibitors (Cammarota et al., 1997). GAP-43 hasbeen proposed to be involved in the generation of activity-dependent synapticmorphological changes (Benowitz & Routtenberg, 1997), a function which maybe important for long-term memory (see below).

The cAMP/PKA/CREB-P Cascade

Like LTP in rat hippocampus (Grant & Silva, 1994; Huang & Kandel, 1995,1996; Huang, Li, & Kandel, 1994), 5HT-induced facilitation in the molluskAplysia (Bartsch et al., 1995), odor conditioning in the fruit fly Drosophila(Tully, 1996; Yin & Tully, 1996), long-term neuromuscular junction plasticityin Drosophila (Davis, Schuster, & Goodman, 1996), spatial learning in themouse (Bourchuladze, Frenguelli, Blendy, Cioffi, Schutz, & Silva, 1994), and,at least in part, inhibitory avoidance in the chick (Rose, 1995a,b; Zhao et al.,1995), the biochemical events triggered by inhibitory avoidance learning in rathippocampus, involve the late intervention of a cyclic adenylyl monophosphate(cAMP)/PKA/CREB (cAMP response element-binding protein) signaling path-way (Bernabeu et al., 1997a–c; Bevilaqua et al., 1997). CREB is a family oftranscription factors that regulate the synthesis of a number of proteins, in-cluding inducible transcription factors, when phosphorylated (CREB-P) (Daviset al., 1996; Ferrer et al., 1996). PKA-mediated CREB-P activation modulatesgene activation and protein synthesis critical for persistence of all the formsof plasticity mentioned above beyond 3 or 4 h (Bourchuladze et al., 1994;Carew, 1996; Deisseroth, Bito, & Tsien, 1996; Martin & Kandel, 1996; Tully,1996; Yin & Tully, 1996).

Like LTP (Frey, Huang, & Kandel, 1993), memory of step-down inhibitoryavoidance is enhanced by the intrahippocampal administration of the 8-Branalog of cAMP or by stimulators of adenylyl cyclase 3 or 6 h after training(Bernabeu et al., 1996, 1997a,c; Bevilaqua et al., 1997).

Hippocampal cAMP levels slowly increase beginning 60 min after step-downinhibitory avoidance training, reaching a peak 180–360 min after training;the increase is not accompanied by changes in the activity of cAMP-specificphosphodiesterase, suggesting that it is due to enhanced adenylyl cyclase activ-ity (Bernabeu et al., 1996, 1997a,b).

PKA activity increases after training, in two peaks: The first, immediatelyafter training, and the second, higher, 3 to 6 h after training (Fig. 4). Thesecond peak correlates with the maximum rise of cAMP levels after trainingand may be triggered by it (Bernabeu et al., 1997a). It is not known whattriggers the earlier peak of PKA activity (Bernabeu et al., 1997b).



FIG. 4. The cAMP/PKA/CREB-P cascade. CA1 levels of cAMP measured by radioimmunoassay(gray squares), PKA activity measured using Kemptide as a substrate (solid squares), and CREB-P (gray squares) and c-fos (tilted crosses) measured by immunohistochemistry, and effect of post-training infusion into CA1 of 8-Br-cAMP (1.25 mg/side; open triangles) and of the PKA inhibitorKT5720 (2.5 mg/side; solid triangles) on retention of the avoidance task. cAMP levels rise slowlyafter training, reached a peak at 180 min, remained high at 360 min, and returned to normal at540 min. There were two peaks of PKA activity and CREB-P: the first 0 min after training, anda second, higher peak at 180–360 min; both PKA values and CREB-P levels returned to normalat 540 min from training. There was a late increase of c-fos levels which accompanied the secondPKA/CREB-P peak. Posttraining 8-Br-cAMP enhanced memory when given 180 or 360 min aftertraining. The PKA inhibitor was amnestic when given 0, 180, or 360 min after training, but not60 or 540 min after training. Data from Bernabeu et al. (1996, 1997b) and Bevilaqua et al. (1997).The data point to a crucial role in memory consolidation of a cAMP/PKA/CREB-P cascade in CA1,accompanied by a c-fos increase, as well as to an early posttraining involvement of PKA (presum-ably using endogenous ‘‘basal’’ cAMP levels) and CREB-P in this process.

There is, thus, a major difference between the PKA cascade and those initi-ated by PKG, PKC, or CaMKII. The PKG cascade lasts less than 30 min; theCaMKII cascade lasts little more than 60 min; the PKC cascade extends forabout 120 min; and the PKA increase occurs in two peaks, the first immediatelyand the second several hours after training (Fig. 4). The data fit with thosethat have been described for hippocampal LTP (see Bliss & Collingridge, 1993;Huang et al., 1994; Huang & Kandel, 1995, 1996; Izquierdo & Medina, 1995;Kim, 1996; Reymann, 1993; Zhuo et al., 1994a,b).

CREB-P levels in the CA1 region also increase after step-down avoidancetraining, in two peaks: One, immediately after training, and another one,larger, 3 to 6 h after training (Bernabeu et al., 1997a,b) (Fig. 4). Both CREP-P peaks correlate with those of increased PKA activity in the hippocampus(Fig. 4). The second ‘‘wave’’ of PKA and CREB-P is also coincident in time



with the amnestic effect of intrahippocampal KT5720 (Bevilaqua et al., 1997;Izquierdo et al., 1997b; Fig. 4).

CREB-P induces synthesis of the transcription factor c-fos, among otherproteins (Ferrer et al., 1996). C-fos increases have been reported not only afterLTP (Dragunow, Abraham, Goulding, Mason, Robertson, & Faull, 1989) butalso, sometimes very rapidly, after a great variety of forms of stimulation(Kaczmarek, 1992). We did in fact observe an increase of c-fos levels 3–6 hafter training in CA1 (Fig. 4), coincident with the second peak of CREB-P.Posttraining c-fos increases are seen in CA2, CA3, entorhinal cortex, and poste-rior parietal cortex, preceded in those cases by a lower early (0 h) peak; no c-fos changes were detected in striatum or thalamus (Bernabeu et al., 1997b).

The first ‘‘wave’’ of CREB-P and c-fos might be related to what Frey andMorris (1997) have recently defined as a ‘‘synaptic tag’’; i.e., a short-lastingmolecular event that sets the stage for the development of a later, protein-dependent phase of LTP.

It is possible that the second ‘‘wave’’ of PKA, CREB-P, and c-fos that occursin the hippocampus after training is functionally more important than thefirst, since it lasts several hours and is modulated by substances acting ondopaminergic, noradrenergic, and 5HT1A receptors (see below).

Several years ago, based upon pioneering findings of his group on two post-training peaks of RNA, protein, and glycoprotein synthesis (see below), oneright after training and the other 5–8 h later, Matthies (1982, 1989) elaboratedon the possible function significance of these two metabolic ‘‘waves,’’ long beforeCREB and its implications for plastic processes were discovered.

A recent experiment by Guzowski and McGaugh (1997) underlines the keyimportance of hippocampal CREB for the persistence of memory for more than4 h (as had been shown for hippocampal LTP; see Huang & Kandel, 1995,1996; Huang et al., 1994). The administration of an oligodeoxynucleotideagainst CREB mRNA into rat hippocampus prior to training hinders memoryof water maze learning measured 48, but not 4, h after training; a random-sequence oligodeoxynucleotide has no effect (Guzowski & McGaugh, 1997).

The Significance of Posttraining Biochemical Events in the Hippocampus

The early events (glutamate receptor activation, etc.) and the PKG, PKC,CaMKII, and cAMP/PKA/CREB-P cascades are sequentially articulated inLTP (Baudry et al., 1996; Bliss & Collingridge, 1993; Deisseroth et al., 1996;Frey et al., 1993; Huang et al., 1994; Izquierdo, 1997; Izquierdo & Medina,1995, 1997; Malinow, 1994; Maren & Baudry, 1995; Martin & Kandel, 1996;Mayford et al., 1996; Reymann, 1993) and maintain glutamatergic transmis-sion enhanced for at least 6 h. It is to be presumed that they subserve a similarrole in the hippocampus in memory consolidation, inasmuch as they representmajor pathways used by the hippocampus to generate long-lasting synapticplasticity of a facilitatory type.

The postulation that memory may rely on LTP (Izquierdo et al., 1992;Lynch & Baudry, 1984; Matthies, 1989) has been very heuristic over the years,in particular, because it led to the demonstration of the main steps involvedin memory formation in the rat hippocampus (see above) and in other formsof plasticity both in the hippocampus and elsewhere (Carew, 1996), which arevery similar to those of the various forms of LTP (Izquierdo & Medina, 1995,1997; Maren & Baudry, 1995).



Discussion of this issue, however, has now become a bit idle (Barnes, 1996).It should be expected that in most situations in which the hippocampus isrequired to maintain synaptic function enhanced for a long time it will usesimilar biochemical processes, as other systems do (see Bartsch et al., 1995;Carew, 1996; Davis et al., 1996; Rose, 1995a,b; Yin & Tully, 1996). In thehippocampus or cerebellum, slight departures from these sequences (for exam-ple, a predominance of phosphatase over CaMKII activity) generate insteadlong-lasting depression (Bindman, Christofi, Murphy, & Nowicki, 1991; Tsu-moto, 1993).

In learning situations, the hippocampus is supposed to receive the stimulirelevant to the training experience from collaterals of the sensory system, fromthe prefrontal cortex, and from the entorhinal cortex (Damasio, 1995; Green,1964; Hyman, van Hoesen & Damasio, 1990; Izquierdo et al., 1992; Willner,Bianchin, Walz, Bueno e Silva, Zanatta, & Izquierdo, 1993; Witter, Groene-wegen, Lopes da Silva, & Lohman, 1989). These stimuli may or may not arrivein volleys such as are needed to provoke clear-cut, long-lasting LTP (Bliss &Collingridge, 1993; Huang et al., 1994). There are now many known types ofLTP, including some that are NMDA-independent, some that need and othersthat do not need the activation of metabotropic receptors, some that involvepresynaptic changes more than others, some that are and others that arenot regulated by retrograde synaptic messengers, some whose late protein-dependent phase is modulated by D1 receptors and others in which this phase ismodulated by b-adrenoceptors, etc. (Bliss & Collingridge, 1993; Collingridge &Bliss, 1995; Huang & Kandel, 1995, 1996; Kleschevnikov, Sokolov, Kuhnt,Dawe, Stephenson, & Voronin, 1997; Malinow, 1994; Nicoll & Malenka, 1995).It is possible, even likely, that the hippocampus will use biochemical sequencessimilar to those of one or another type of LTP depending on the task and onthe subarea that plays a primary role (CA3 or CA1, the subiculum, etc.; seeGabrielli et al., 1997; Mayford et al., 1996).

At this stage, it is wise to simply admit that the biochemical events ofmemory formation in rat hippocampus are very much like those of LTP (Berna-beu et al., 1997a,b; Bevilaqua et al., 1997; Carew, 1996; Izquierdo & Medina,1995, 1997; Figs. 1–4) and to refrain from proposing causal relationships be-tween the two sets of phenomena. That there may in many cases be an overlap,and, for example, repetitive aversive training may block CA1 LTP (Izaki &Arita, 1996), is the least that can be expected from processes that share thesame cell population and many of the same mechanisms. However, many such‘‘saturation’’ studies have failed (see Barnes, 1996), even when successive LTPswere used in the same neuron field (Frey, Schollmeier, Reymann, & Seiden-becher, 1995), let alone when the effect of learning on LTP or vice versa wasinvestigated (Barnes, 1996; Bliss & Richter-Levin, 1993).

RELATION OF HIPPOCAMPAL EVENTS WITH THOSEIN OTHER REGIONS OF THE BRAIN

As mentioned above, some of the early events of memory formation in thehippocampus also occur in amygdala, medial septum, and entorhinal cortex:Early sensitivity to NMDA and AMPA receptor blockers, GABAA receptor ago-nists, cholinergic muscarinic and noradrenergic agonists and antagonists, andPKC or CaMKII inhibitors (Davis, 1992; Izquierdo et al., 1992; Jerusalinskyet al., 1992, 1993; Jerusalinsky, Quillfeldt, Walz, Da Silva, Bueno e Silva,



Bianchin, Zanatta, Ruschel, Schmitz, Paczko, Medina, & Izquierdo, 1994a;Wolfman et al., 1994). The late cAMP/PKA-dependent phase is absent in theamygdala (Bevilaqua et al., 1997). Most of the biochemical changes listed abovehave been observed in CA1, but some have been observed in other hippocampalsubareas as well (see above). This should not be surprising, given the directsynaptic connection between CA3 and CA1 (Hyman et al., 1990), and thediverse indirect connections that exist among the various hippocampal subre-gions (Green, 1964; Iijima, Witter, Ichikawa, Tominaga, Kajiwara, & Matsu-moto, 1996; Witter et al., 1989).

It is possible (Jerusalinsky et al., 1992, 1994a) that short or abortive formsof LTP-like phenomena (Bliss & Collingridge, 1993; Reymann, 1993) may par-ticipate in early posttraining memory processing by the amygdala or medialseptum. Involvement of these areas depends on the task. In inhibitory avoid-ance all of them are necessary; in less emotional tasks, like habituation to anovel environment, the amygdala and the septum are not involved (Izquierdoet al., 1992; Izquierdo & Medina, 1991, 1995; Wolfman et al., 1991); in a highlyemotional task (conditioned fear), the amygdala plays a major role (Davis,1992) and apparently this role includes CaMKII (Mayford et al., 1996), as itdoes in inhibitory avoidance (Wolfman et al., 1994).

The timing of the onset of the initial NMDA-dependent, muscimol-dependentphase of memory in hippocampus (and amygdala), entorhinal, and parietalcortex is sequential, which indicates that these structures operate sequentiallyand concertedly in the formation of memories (Izquierdo, Quillfeldt, Zanatta,Quevedo, Schaeffer, Schmitz, & Medina, 1997a; Zanatta, Schaeffer, Schmitz,Medina, Quevado, Quillfeldt, & Izquierdo, 1996). This phase extends for a fewminutes after training in hippocampus and amygdala, starts 30 min aftertraining in entorhinal cortex, and 60 min after training in posterior parietalcortex. The time in which memory is sensitive to the amnestic effect of AP5or muscimol is less than 30 min in hippocampus or amygdala, but lasts up to270 min in the entorhinal or parietal cortex (Zanatta et al., 1996). The possiblepathways involved should be searched for among those reviewed by Hymanet al. (1990) and Witter et al. (1989).

In addition to its late role in memory processing, other data point to an earlyintervention of the entorhinal cortex in learning, as a station through whichsignals eventually reach the hippocampus so that this can play its role inconsolidation (Squire, 1992; Jerusalinsky et al., 1994a; Willner et al., 1993).

MODULATORY INFLUENCES IN THE IMMEDIATEPOSTTRAINING PERIOD

The early events (õ30 min after training) are subject to inhibition by GABAA

receptors (Brioni, 1993; Izquierdo, 1997; Izquierdo et al., 1992, 1997a) modu-lated by endogenous benzodiazepine-like substances (Izquierdo & Medina,1991; Wolfman et al., 1991) and are, in addition, modulated by cholinergicmuscarinic and b-noradrenergic synapses (Izquierdo et al., 1992).

A variety of central and peripheral modulatory systems also influence mem-ory in the early posttraining period: b-Endorphin, cholinergic nicotinic recep-tors, serotonin, adrenocorticotropin, vasopressin, oxytocin, glucocorticoids, epi-nephrine, norepinephrine, and glucose (see Bohus, 1994; Brioni, 1993; Gold,1995; Izquierdo, 1989; Izquierdo & Medina, 1995, 1997; McGaugh, 1989;McGaugh, Cahill, Parent, Mesches, Coleman-Mesches, & Salinas, 1995; Roo-



zendaal & McGaugh, 1996). Most of these substances are in fact releasedduring many forms of behavioral training, which signifies that many or mostmemories are actually formed and often retrieved while under their influence(Izquierdo, 1989). Corticosterone also modulates memory consolidation of in-hibitory avoidance in the chick (Rose, 1995a).

All these substances affect hippocampal LTP (see Gold, 1995; Izquierdo &Medina, 1997). Cholinergic muscarinic agents upregulate NMDA receptors,activate the phosphoinositide cascade, and alter protein kinase C activity (seeJerusalinsky et al., 1997). Catecholamines enhance cAMP levels and therebyprotein kinase A activity (Bevilaqua et al., 1997). Peripherally administeredcatecholamines influence central catecholamine levels and actions (Gold, 1995;McGaugh et al., 1995), vasopressin and corticotropin influence brain catechol-amine levels (see Bohus, 1994; Gold, 1995, for references), and corticotropin,corticosteroids, and opioid agonists and antagonists influence brain norepi-nephrine effects (see Izquierdo, 1989; McGaugh et al., 1995).

Most of these agents act primarily through interactions with brain noradren-ergic mechanisms in the amygdala (Cahill & McGaugh, 1996; Gallagher, Kapp,Musty, & Driscoll, 1977; McGaugh, 1989; McGaugh & Cahill, 1997; McGaughet al., 1995; McGaugh, Introini-Collison, Juler, & Izquierdo, 1986). Their maininfluence on the hippocampal mechanisms of memory formation is thereforeindirect and must consist of interferences with direct or indirect amygdala–hippocampus connections (Hyman et al., 1990; Witter et al., 1989) early aftertraining. Peripherally administered opioids and opioid antagonists alter mem-ory also partly through actions on the medial septum (Bostock, Gallagher, &King, 1988; Izquierdo, 1989), which is of course directly linked to the hippocam-pus (Gray, 1982). Glucocorticoids may also act through specific receptors inhippocampus (De Kloet, Rosenfeld, Van Eekelen, Ratka, Joels, & Levine, 1989).Peripheral epinephrine, norepinephrine, and corticoids regulate glycemia, andhyperglycemia mimics the memory-facilitatory effects of these substances andenhances hippocampal LTP (Gold, 1995).

It is generally agreed that the amygdala is the main brain center that addsemotional ‘‘colors’’ or ‘‘tinges’’ to memories at the time of encoding (Cahill &McGaugh, 1996; Gallagher et al., 1977; McGaugh & Cahill, 1997; McGaugh etal., 1995) and at the time of retrieval (see Bechara, Tranel, Damasio, Adolphs,Rockland, & Damasio, 1995; Izquierdo, 1989; Izquierdo et al., 1997a; Rogan &Le Doux, 1996; Scott, Young, Calder, Hellawell, Aggleton, & Johnson, 1997).Some evidence suggests that the medial septum/diagonal band complex mayplay a similar role (see Cahill & McGaugh, 1996; Gray, 1982; Heimer, Harlan,Alheid, Garcia, & De Olmos, 1997; Izquierdo et al., 1992; Wolfman et al., 1991).Throughout onto- or phylogeny, the corticomedial prefrontal cortex may takeover some of these functions (Damasio, 1995).

Despite earlier claims (e.g., Bianchin, Walz, Ruschel, Zanatta, Da Silva,Bueno e Silva, Paczko, Medina, & Izquierdo, 1993), most current evidenceindicates that the amygdala is not a site of storage (Bevilaqua et al., 1997;Izquierdo et al., 1997; McGaugh & Cahill, 1997; McGaugh et al., 1995; seebelow). The same may be said of the medial septum/diagonal band area. Thisstructure is believed to influence memory through its direct connection withthe hippocampus (Izquierdo & Medina, 1997), and this connection has a time-limited role in memory formation (Kim, Clark, & Thompson, 1995; Cahill &McGaugh, 1996).

Hormones and neuromodulators have been proposed to alter memory by



direct influences on gene transcription (Sossin, 1996). However, corticotropin,glucocorticoids, vasopressin, epinephrine, norepinephrine, and catecholaminereleasers facilitate memory at low doses but impair memory at high doses(Gold, 1995; Izquierdo, 1989; McGaugh et al., 1995). This argues against adirect role on gene transcription, which should presumably follow a lineardose–response curve. It is known that low to moderate levels of arousal, or mildanxiety, facilitate learning and retrieval, whereas too high levels of arousal oranxiety have an impairing effect (see Gold, 1995, for references).

Chronic stress has been repeatedly implicated in the genesis of depression(Post, 1992; Willner, 1990) and, of course, of posttraumatic stress disorder,both of which have profound cognitive influences. Chronic stress or repeatedglucocorticoid release or administration damages hippocampal pyramidal cells(see McEwen & Sapolsky, 1995). The role of brain benzodiazepine-like sub-stances in memory modulation may be related to the regulation of anxiety orstress (Izquierdo & Medina, 1991; Wolfman et al., 1991). Korneyev (1997) hasproposed that memory-related effects of anxiolytic drugs may be mediated byinfluences on the hypothalamic–pituitary–adrenocortical axis.

MODULATORY INFLUENCES 3 TO 6 H AFTER TRAINING

In the rat, the late events in the hippocampus (3.0–6.0 h after training) aremodulated by dopamine D1, b-noradrenergic, and 5HT1A receptors (Bevilaquaet al., 1997). Infusion into CA1 of the D1 agonist SKF38393, of the b agonistnorepinephrine, or of the 5HT1A antagonist NAN-190 3 or 6 (but not 0, 1.5, or9) h after training strongly facilitates memory of the step-down task. Infusion 3or 6 h after training of the PKA inhibitor KT5720 (Fig. 4), of the D1 antagonistSCH23390, of the b antagonist timolol, or of the 5HT1A antagonist 5-HO-DPAT is instead strongly amnestic. D1 and b-noradrenergic receptors areknown to stimulate, and 5HT1A receptors to depress, adenylyl cyclase activity.Their late modulation of memory seems indeed linked to these effects. For-skolin, another adenylyl cyclase activator, and 8-Br-cAMP itself enhance mem-ory when infused into CA1 3 or 6 (but not 0, 0.5, or 9) h after training (Bernabeuet al., 1996; Bevilaqua et al., 1997; Fig. 4).

These findings indeed correlate with the increase of the levels of hippocampalcAMP (Bernabeu et al., 1996) and CREB-P (Bernabeu et al., 1997a,b) com-mented on above (Fig. 4). They also agree with findings from Kandel’s groupof a late (i.e.,ú3 h) upregulation of CA1 LTP by dopamine D1 receptors (Huanget al., 1994) and of CA3 LTP by b-noradrenergic receptors (Huang & Kandel,1996). 5HT1A receptors downregulate the early phase of dentate gyrus LTP(Sakai & Tanaka, 1993).

Dopamine D1, b-noradrenergic, and 5HT1A receptors are of course activatedby the dopaminergic, noradrenergic, and serotoninergic pathways which arebelieved to play a key role in the regulation of mood and affect (see Heimeret al., 1997; Izquierdo & Chaves, 1996; Willner, 1990). Thus, the memorydisturbances seen in disorders of these systems (e.g., schizophrenia, depres-sion, Parkinson’s disease; Izquierdo & Chaves, 1996) may be related to alter-ations in the regulation of the late phase of memory formation. It must benoted that the late time window during which memory formation is exposedto the influence of dopamine D1, b-noradrenergic, and 5HT1A receptors in thehippocampus is very wide (ú3 h) (Bevilaqua et al., 1997). Recent unpublishedfindings from our laboratory suggest that a similar, parallel sensitivity to D1,



b and 5HT1A receptors, forskolin, and 8-Br-cAMP exists in the entorhinalcortex in relation to memory (see Bevilaqua et al., 1997).

Corticosteroid modulation of memory formation in the chick brain has beenattributed to an influence on the late synthesis of glycoproteins (Rose, 1995a).

Dopaminergic, noradrenergic, and serotoninergic systems, by directly affect-ing the biochemical processes of memory, introduce information of their own,adding components (Izquierdo et al., 1992; Izquierdo & Medina, 1991, 1995),‘‘colors’’ or ‘‘tinges,’’ to the memories, pertaining to alerting, emotional or af-fective components (Bohus, 1994; Cahill & McGaugh, 1996; Izquierdo & Me-dina, 1991, 1995, 1997). In other words, the biochemical cascades in a givenset of synapses in the hippocampus may change depending on direct or indirectinfluences of the various neuromodulators and hormones on this structure(Izquierdo & Medina, 1997). The cognitive influence of mood, affect, and emo-tion has been known for years (see below and Bohus, 1994; Izquierdo & Medina,1995; Rogan & Le Doux, 1996).

SIMILAR BIOCHEMICAL EVENTS IN CHICK BRAIN

Many of the biochemical steps described above have been also observedin the intermediate medial hyperstriatum ventrale (IMHV) and in the lobusparaolfactorius (LPO) of the chick brain after another form of inhibitory avoid-ance (not to peck a bitter bead) (Rose, 1995a,b). In the chick, the data showearly upregulation of NMDA receptors in the IMHV (Stewart, Bourne, &Steele, 1992), an amnestic effect of NMDA receptor antagonists (Bourchu-ladze & Rose, 1992), an early role for metabotropic receptors (Rickard & Ng,1995), increased CaMKII activity (Zhao et al., 1995), early phosphorylation ofGAP-43 coupled with increased PKC activity (Bourchuladze et al., 1990), andboth an early and a late activation of early genes, signaled by increases of c-fos and c-jun (Anokhin et al., 1991). A major difference between the biochemicalchanges reported in hippocampus and those found in the chick brain is theapparent lack of participation of AMPA receptors in the latter (Bourchuladze &Rose, 1992; Rose, 1995b).

The sequential participation of the hippocampus, entorhinal cortex, and pari-etal cortex in memory consolidation in rats is remindful of what happens inthe chick, where the IMHV and the LPO enter into play sequentially aftertraining in inhibitory avoidance (Rose, 1995b).

Further, and important, similarities between posttraining biochemicalevents in rat hippocampus and in chick brain exist at the level of late changesin synaptic glycoproteins (see below).

ROLE OF THE HIPPOCAMPUS AND OTHERSTRUCTURES IN RETRIEVAL

The involvement of the hippocampus in many if not all forms of declarativememory has been known for a long time (Barnes, 1979, 1996; Barnes & Mc-Naughton, 1985; Corkin, Cohen, Sullivan, Clegg, Rosen, & Ackerman, 1985;Eichenbaum, 1996; Green, 1964; Milner, 1970; Squire, 1992).

The hippocampus forms part of three related but different circuits: One atthe time of acquisition, another at the time of memory formation, and anotherat the time of retrieval.

During acquisition it must receive information from the working memory/



manager regions of the prefrontal cortex (Fuster, 1995), which is related bythe entorhinal cortex and the dentate gyrus (Hyman et al., 1990; Willner etal., 1993; Witter et al., 1989). Acquisition is not the concern of the presentarticle.

In the posttraining period of inhibitory avoidance, the hippocampus oper-ates concertedly with the amygdala and medial septum/diagonal band area(Izquierdo et al., 1992) and, 30 to 60 min later, with the entorhinal andparietal cortex (Izquierdo et al., 1997a; Zanatta et al., 1996) (see above). Inhumans, possibly different hippocampal subregions participate in the encod-ing and retrieval of different tasks (Gabrielli et al., 1997). Brioni (1993) hasconsidered the fact that the various findings on the simultaneous and coordi-nated activity in the posttraining period are evidence for ‘‘the multiple consoli-dation of memory.’’

At the time of retrieval, in the first few days after training, the hippocampusand amygdala integrate a network with the entorhinal and parietal cortex, allof which are needed for retrieval (Izquierdo et al., 1997a; Quillfeldt, Zanatta,Schmitz, Quevado, Schaeffer, De Lima, Medina, & Izquierdo, 1996). Their rolerequires AMPA receptors: CNQX infused prior to testing into any of thesestructures causes a transient block of memory expression. This does not neces-sarily mean that memories are stored in any of these structures, as was erron-eously interpreted some years ago (e.g., Bianchin et al., 1993). It just meansthat the structures are necessary for retrieval; some of them, for example, theamygdala, may be so in order to add emotional or aversive ‘‘tinges’’ to thememory at that time (see Bechara et al., 1995; Scott et al., 1997).

Thirty days after training, pretest CNQX given into the hippocampus andamygdala is no longer effective, but it still hinders retrieval when infused intothe entorhinal or posterior parietal cortex (Izquierdo et al., 1997a; Quillfeldtet al., 1996). This indicates that, by that time, the hippocampus and amygdalaare no longer necessary for retrieval; in fact, some evidence suggests that theemotional functions of the latter could be taken over by the corticomedialprefrontal cortex (Damasio, 1995). This also indicates that the control of mem-ory expression is transferred to the entorhinal and the posterior parietal cortexat 30 days from training (Izquierdo et al., 1997a) and fits with clinical dataon the limited duration of the retrograde amnesia of patients with medialbilateral lesions of the temporal lobe (Corkin et al., 1985; Milner, 1970).

At 60 days from training, pretest CNQX has no effect on retention testperformance when given into the entorhinal cortex, but it is amnestic wheninfused into the parietal area. This shows that, by that time, the posteriorparietal cortex still holds control over memory expression (Izquierdo et al.,1997a). It remains to be seen whether other areas of the brain take over therole of the amygdala (see above) or of the hippocampus and entorhinal cortexat 2 months from the original training experience. Preliminary findings fromthis laboratory show that 90 days after training the effect of pretest CNQXgiven into the parietal cortex is only partial.

It is interesting that the sequence by which these four structures enter intoplays in the process of memory formation is the same in which they disappearfrom the stage at the time of retrieval, although of course with a very differenttime course: First hippocampus and amygdala, then entorhinal cortex, andfinally parietal cortex (Izquierdo et al., 1997a; Quillfeldt et al., 1996; Zanattaet al., 1996).

Possibly, other cortical areas (frontal, cingulate, occipital) also participate in



long-term storage (Bontempi, Jaffard, & Destrade, 1996; Valenstein, Bowers,Verfaellie, Heilman, Day, & Watson, 1987). Some aspects of spatial learninginvolve non-NMDA-mediated mechanisms, probably in the hippocampus(Bannerman, Good, Butcher, Ramsay, & Morris, 1995); other aspects of spatialand nonspatial declarative learning involve the striatum (Packard &McGaugh, 1992).

THE CONNECTION BETWEEN POPSTRAINING HIPPOCAMPALEVENTS AND EFFECTIVE MEMORY CONSOLIDATION:

CELL ADHESION AND SYNAPTIC GROWTH

The biochemical changes seen in the hippocampus after inhibitory avoidancelearning are very large: 20–120% increases of the activity of enzymes suchas NO synthase, heme oxygenase, PKG, PKA, PKC, or CaMKII, several-foldincreases of cGMP and cAMP, 40–80% increases in CREB-P, 20–80% in-creases in AMPA binding or in the amount of measurable GluR1 or NMDA1,etc. (Bernabeu et al., 1996, 1997a,b; Cammarota et al., 1995, 1996, 1997) (Figs.1–4). This precludes any idea that they may be synapse-specific; they are,instead, cellular changes seen over the whole hippocampus or entire subregionsof the hippocampus. Some of the biochemical changes are, no doubt, structure-specific (they are not seen in other brain regions), and all appear to be learning-specific (they are not seen in animals exposed to the footshock alone or tothe apparatus without the footshock). Some consequences of the biochemicalchanges, however, may well be synapse-specific, if they preferentially affectthe synapses that are being activated at the time. Several data suggest thatmany of these changes are indeed activity-dependent (see Izquierdo & Medina,1995, 1997; Rose, 1995a,b).

Common sense dictates that long-term memory storage does not rely on theproduction of new proteins or other molecules, but must involve long-lastingfunctional alterations that make some synapses more efficient and, perhaps,others less efficient (Dunn, 1980). The need for the suppression of some behav-iors in order to permit the acquisition and storage of new ones using the samebrain systems is not given the attention it deserves, despite the fact thatinhibitory avoidance, by description, involves precisely this (Netto & Iz-quierdo, 1985).

Functional synaptic alterations, like those that occur during brain develop-ment, may in many cases rely on anatomically visible changes. Three decadesago, Bennett, Diamond, Krech, and Rosenzweig (1964) reported pronouncedchanges in brain weight and protein content in rats raised in an enrichedenvironment. Many studies in animals and in rats have borne out these semi-nal findings, including clinical and experimental observations on the recoveryof function after hemispherectomy at an early age (see Izquierdo, 1997), orafter fornix lesions in rats (van Rijzingen, Gispen, & Spruijt, 1997), which canonly be explained by extensive synaptic growth and rearrangement in theremaining brain tissue.

Ramon y Cajal (1952), early in this century, was the first to propose thatmorphological changes underlie plastic phenomena in synapses. Growth(Greenough, Hwang, & Gorman, 1985), shortening (Brandon & Coss, 1982),and an increase in number (Patel & Stewart, 1988) and/or a loss (Wallhaus-ser & Scheich, 1987) of dendritic spines or other components of synapses havebeen observed in the hippocampus and elsewhere a few hours after brief train-



ing experiences. It is very likely that more subtle changes in synaptic morphol-ogy may occur in many, if not most, forms of long-term plasticity (Benowitz &Routtenberg, 1997). For example, Geinisman, de Toledo-Morrell, Morrell,Heller, Rossi, and Parshall (1993) have described the development of complete,multiple partitioning of transmission zones in axospinous synapses of the hip-pocampus following repeated LTP. Such partitioning would escape notice un-less very specific neuroanatomical methods are used.

Very recently, a 25- to 30-fold increase in hyperchromic granule cells (tolu-idine blue staining) has been observed 5 to 7 h after inhibitory avoidancetraining in the adult rat hippocampus; the cells group in well-defined ribbon-like clusters and present a large increase in spine numbers (O’Connell et al.,1997). It is tempting to relate these changes to the biochemical events thatoccur in the hippocampus precisely at that time (CREB-P increase, c-fos syn-thesis, see above). The dentate gyrus is, of course, not only an input linkbetween the entorhinal cortex and the hippocampus proper, but also a majorsite of projection from the hippocampus, as are the amygdala, entorhinal cor-tex, and septum (Hyman et al., 1990; Iijima et al., 1996; Witter et al., 1989).

Activity-dependent changes in cell adhesion explain changes in synapticfunction (Field & Itoh, 1996; Rose, 1995a). Glycoproteins in the external sideof cell membranes play a major role in this (Rose, 1995a). Increased fucoseuptake occurs in two ‘‘waves’’ after brightness discrimination training, in bothhippocampus and neocortex: Early after training, and again 7 or 8 h later(Matthies, 1982; Pohle, Ruthrich, Popov, & Matthies, 1979). Two similarlyspaced ‘‘waves’’ of sensitivity to the amnestic effect of locally injected antago-nists of terminal fucosylation of glycoproteins have been described in the chickbrain (Rose, 1995a; Rose & Jork, 1987; Scholey, Rose, Zamani, Bock, &Schachner, 1993) and in the rat hippocampus (Doyle, Nolan, Bell, & Regan,1992a) following different forms of one-trial inhibitory avoidance training. Theenhanced clustering of hyperchromic dentate granule cells 5 or 7 h after inhibi-tory avoidance shown by O’Connell et al. (1997) has been ascribed to changesin cell adhesion factors, such as the L1 and the neural cell adhesion molecules(NCAMs). L1 and NCAMs have been attributed a regulatory role in the mainte-nance of hippocampal CA1 LTP (Field & Itoh, 1996; Luthi, Laurent, Figurov,Muller, & Schachner, 1994). Antibodies to these substances hinder memory ofinhibitory avoidance when given before or 5.5–8 h after training into the ratcerebral ventricles (Doyle, Nolan, Bell, & Regan, 1992b) or into the chick brain(Scholey, Mileusnic, Schachner, & Rose, 1995). Importantly, an antibody raisedagainst chick forebrain NCAM is amnestic not only for inhibitory avoidancein the chick, but also when injected intracerebroventricularly into rats 5.5 hafter training, for inhibitory avoidance in the rat (Alexinsky, Przybyslawski,Mileusnik, Rose, & Sara, 1997).

It is interesting to note that the second ‘‘wave’’ of changes related to theglycoprotein matrix, or in some cases synaptic morphologic changes, followsthe cAMP/PKA/CREB-P/protein synthesis changes by 1 or 2 h, which is coher-ent with the idea that they may be a consequence of the latter (Matthies, 1982,1989; see Field & Itoh, 1996; Rose, 1995a).

A role for the GAP-43 protein in synaptic morphological changes has beensuggested by Benowitz and Routtenberg (1997). As mentioned above, we haverecently shown PKC-mediated phosphorylation of hippocampal GAP-43 30 minafter inhibitory avoidance training in the rat (Cammarota et al., 1997; Fig. 3).

The morphological changes seen in the dentate gyrus or elsewhere may also



be related to expression of the gene for extracellular serine protease tissueplasminogen activator (tPA), which apparently supports, alongside withCREB, the prolongation of LTP beyond 4 h in CA1 (Frey, Muller, & Kuhl,1996). The release of tPA is linked to morphological differentiation and maycorrelate with activity-dependent morphological changes (see above). DuringLTP and other forms of enhanced electrical activity tPA is induced throughan NMDA-receptor-mediated mechanism in rat hippocampus as an immediateearly gene (Qian, Gilbert, Colicos, Kandel, & Kuhl, 1993).

There is no evidence that memory is stored in the form of LTP. This couldbe a possibility in the case of the hippocampus, where LTPs lasting for severalweeks have indeed been described (Barnes, 1979; Barnes & McNaughton,1985) and where interference with the expression of AMPA receptors at thetime of testing also hinders retrieval for a few weeks (Bianchin et al., 1993;Izquierdo et al., 1997a; Quillfeldt et al., 1996). For memories persisting morethan a few weeks in whose retrieval cortical structures other than the hippo-campus play a role (entorhinal cortex, posterior parietal cortex, etc.; see above),certainly it might be wiser to think of mechanisms different from LTP, suchas activity-dependent synaptic adhesion changes mediated by glycoproteins,followed by morphological changes (Rose, 1995a,b).

AN ACTIVE LOOP BETWEEN THE HIPPOCAMPUSAND MODULATORY SYSTEMS

One point deserves comment as a possible source of further study. Activa-tion of dopaminergic, noradrenergic, and serotoninergic pathways around 3h after training appears to occur physiologically: The blockers SKF 23390,timolol, and NAN-190 had powerful actions on their own when infused intothe hippocampus but not the amygdala at that time or later (Bevilaqua et al.,1997). Therefore, there must be hippocampofugal pathways that inform thesubstantia nigra, the locus coeruleus, and the raphe nuclei, i.e., the siteswhere the cell bodies of the brain dopaminergic, noradrenergic, and serotonin-ergic systems are (Nieuwenhuys, 1985), that a learning situation has takenplace 3 h before. A variety of such pathways exists (Nieuwenhuys, 1985;Witter et al., 1989).

On the basis of the known influence of D1, b, and 5HT1A receptors onadenylyl cyclase, the message back from these areas to the hippocampus (butnot the amygdala) is quite clear. Two pathways, the one ending on D1 receptorsand the one ending on b-adrenoceptors, instruct the hippocampus to producemore cAMP and so activate the PKA/CREB-P pathway, induce protein synthe-sis, and save the learned information. The pathway that innervates the 5HT1Areceptors obviously gives a contrary message.

CONCLUSION AND A WORKING HYPOTHESIS

To summarize, in the first 6 h after training there is a chain of biochemicalevents in the hippocampus that is necessary for memory processing. There aretwo ‘‘waves’’ in this chain: One right after training, and the other, 3–6 h later,both related to cAMP-sensitive gene transcription. Each of these ‘‘waves’’ isfollowed by changes in glycoprotein synthesis and by events that may be ex-plained by changes in cell adhesion, particularly the second ‘‘wave.’’ It is likelythat the second wave is triggered by the reflex activation of noradrenergic and



dopaminergic afferents to the hippocampus and controlled by serotoninergicfibers.

At the time of training and for a short period afterward, the amygdala andperhaps the medial septum contribute to memory processing by bringing inemotional information. Later, the entorhinal cortex and then the posteriorparietal cortex become involved and become necessary both for memory forma-tion and for long-term retrieval.

It is likely that electrical activity originated in the hippocampus may regu-late cell adhesion changes over the several hours that follow after trainingin the hippocampus itself, the dentate gyrus, and the neocortex. This coulddetermine synapse specificity with the need of anything but housekeepingmetabolic activities of nerve cells, based on the chain of events that goesfrom glutamate receptor activation to gene transcription (Field & Itoh, 1996;Rose, 1995a). This hypothesis may be traced back to Matthies (1982, 1989)or Rose (1995).

A device must be postulated in order to sustain activity-dependent cell adhe-sion changes for long periods of time, at least until synaptic morphologicalchanges have become established. One is the replay of learning-related activityby hippocampal cells that project to those synapses. A replay of correlatedneuronal firing patterns acquired during spatial learning by groups of hippo-campal pyramidal cells has been recently reported to occur in rats during sleep(Skaggs & McNaughton, 1996). The need for such an activity is suggested bythe well-known fact that patients with medial temporal lesions have a retro-grade amnesia of months (Corkin et al., 1985; Milner, 1970), whereas the directinvolvement of that region in storage processes is of course much shorter (seeIzquierdo et al., 1997a).

Experiments are in progress in order to determine the links between thehippocampal chain of events in inhibitory avoidance in rats and activity-depen-dent changes in cell adhesion properties, as well as between the hippocampusand the modulatory neuronal systems of the brain.

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