Histamine in the basolateral amygdala promotes PNAS PLUS inhibitory avoidance learning independently of hippocampus

Fernando Benettia,1, Cristiane Regina Guerino Furinia, Jociane de Carvalho Myskiwa, Gustavo Provensib, Maria Beatrice Passanib, Elisabetta Baldic, Corrado Bucherellic, Leonardo Munarib,2, Ivan Izquierdoa,3, and Patrizio Blandinab,3

aMemory Center, Brain Institute of , Pontifical Catholic University of Rio Grande do Sul, 90610-000 , RS, Brazil; bDipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmacologia e Tossicologia, Universitá di Firenze, 50139 Firenze, Italy; and cDipartimento di Medicina Sperimentale e Clinica, Universitá di Firenze, 50134 Firenze, Italy

Contributed by Ivan Izquierdo, March 30, 2015 (sent for review March 4, 2015; reviewed by Federico Bermudez-Rattoni and Daniele Piomelli) Recent discoveries demonstrated that recruitment of alternative hippocampal damage before new learning can be overcome, possibly brain circuits permits compensation of impairments follow- through recruitment of an alternative circuit. The identity of the ing damage to brain regions specialized in integrating and/or storing compensatory structures is still unknown, but the BLA could specific , including both dorsal hippocampus and baso- potentially be one, because it operates to a certain extent in lateral amygdala (BLA). Here, we first report that the integrity of the parallel with the CA1 region in memory processing (9). brain histaminergic system is necessary for long-term, but not for Extensive evidence indicates that emotionally significant experi- short-term memory of step-down inhibitory avoidance (IA). Second, ences activate many hormones and neurotransmitters, including we found that phosphorylation of cyclic adenosine monophosphate histamine, that regulate the consolidation of newly acquired mem- (cAMP) responsive-element-binding protein, a crucial mediator in ories (10, 11). Histamine is synthesized from histidine by histidine- long-term memory formation, correlated anatomically and tempo- decarboxylase (HDC) (12) and released in the brain from varicos- rally with histamine-induced memory retrieval, showing the active

ities of axons that ramify extensively throughout the central nervous involvement of histamine function in CA1 and BLA in different system. The only source of histaminergic fibers is the hypothalamic phases of . Third, we found that exogenous tuberomamillary nucleus (TMN) (13). The histaminergic system is application of histamine in either hippocampal CA1 or BLA of brain crucial in the sleep–wake cycle and is implicated in various brain histamine-depleted rats, hence amnesic, restored long-term memory; however, the time frame of memory rescue was different for the functions, including the modulation of hippocampal synaptic plas- two brain structures, short lived (immediately posttraining) for BLA, ticity (12, 14). Interestingly, when infused into the CA1 region im- long lasting (up to 6 h) for the CA1. Moreover, long-term memory mediately after training of an IA task, histamine induced a dose- was formed immediately after training restoring of histamine trans- dependent promnesic effect through activation of H2 receptors mission only in the BLA. These findings reveal the essential role of without altering locomotor activity, exploratory behavior, anxiety histaminergic neurotransmission to provide the brain with the plas- ticity necessary to ensure memorization of emotionally salient events, Significance through recruitment of alternative circuits. Hence, our findings indi- cate that the histaminergic system comprises parallel, coordinated Integrity of the brain histaminergic system is necessary for long- pathways that provide compensatory plasticity when one brain term memory (LTM) but not short-term memory of step-down structure is compromised. inhibitory avoidance (IA). Histamine depletion in hippocampus or basolateral amygdala (BLA) impairs LTM of that task. Histamine histamine | hippocampus | amygdala | lateral ventricle | inhibitory infusion into either structure restores LTM in histamine-depleted avoidance rats. The restoring effect in BLA occurs even when hippocampal activity was impaired. Cyclic adenosine monophosphate (cAMP) motionally arousing experiences create long-term memories responsive-element-binding protein phosphorylation correlates Ethat are initially labile, but over time become insensitive to anatomically and temporally with histamine-induced memory disruption through a process known as consolidation (1). One- . Thus, histaminergic neurotransmission appears critical to trial fear-motivated learning tasks, such as step-down inhibitory provide the brain with the plasticity necessary for IA through re- avoidance (IA), a hippocampal-dependent associative learning cruitment of alternative circuits. Our findings indicate that the (2), have largely contributed to the knowledge of consolidation histaminergic system comprises parallel, coordinated pathways process, and convincing evidence indicates that the CA1 region that provide compensatory plasticity when one brain structure is of the hippocampus, the basolateral amygdala (BLA) and the compromised. medial prefrontal cortex, are crucially involved in this process (3, 4). However, the actual contribution of each region remains Author contributions: F.B., C.R.G.F., J.d.C.M., G.P., M.B.P., I.I., and P.B. designed research; F.B., C.R.G.F., J.d.C.M., G.P., E.B., C.B., and L.M. performed research; F.B., C.R.G.F., J.d.C.M., poorly characterized. For instance, it is suggested that they are G.P., I.I., and P.B. analyzed data; and J.d.C.M., M.B.P., I.I., and P.B. wrote the paper. part of mostly independent circuitries specialized in Reviewers: F.B.-R., National University of Mexico; and D.P., University of California, Irvine. specific aspects of information, e.g., the emotional component in the BLA and the cognitive aspect in the hippocampus (2, 5). If The authors declare no conflict of interest. 1Present address: Departamento de Fisiologia, Instituto de Ciências Básicas da Saúde, this relation between structures holds true for an intact brain, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 90050-17, Brazil. however, examples of learning recovery of individuals bearing 2Present address: Department of and Systems Therapeutics, Mount Sinai large brain lesions suggest that the brain can adapt dynamically, School of Medicine, New York, NY 10029. and interconnected systems can provide compensation for selec- 3To whom correspondence may be addressed. Email: [email protected] or patrizio. tive damage (2). Indeed, hippocampal damage resulted in retro- [email protected]. grade for acquired fear memories (6), but did not prevent This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. new learning in rodents (7) as well as in humans (8). Therefore, 1073/pnas.1506109112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1506109112 PNAS Early Edition | 1of7 Downloaded by guest on October 1, 2021 state, or retrieval of the avoidance response (15). Consistently, post- mediator in the formation of long-term memory (21), and an in- training injections into the dorsal hippocampus of histamine H2 or crease in CREB phosphorylation at Ser-133 in the hippocampus is H3 agonists improved memory consolidation after specifically associated with IA memory formation (22, 23). In par- contextual fear conditioning through a mechanism involving extra- ticular, a significant increase of pCREB in the hippocampus occurs cellular signal-regulated kinase (ERK)2 phosphorylation (16). Several immediately after training, followed by a delayed increase of Ser- neurotransmitters, such as , glutamate, and norepineph- 133 pCREB 3–6 h later (22). It is conceivable that for a short period rine, activate the ERK cascade in the hippocampus (17), and hista- after training, the hippocampus acts in concert with the amygdala mine may interact with these neurotransmitters to orchestrate ERK2 that contributes emotional values (2). phosphorylation that appears to play a critical role in consolidating To examine the influences of histamine depletion on CREB emotional memories (17). Histamine modulates memory of emo- phosphorylation following IA training, rats were euthanized tionally arousing experiences also in the BLA. Administration of H3 10 min or 5 h after training, and pCREB levels were assessed in the receptor antagonists into the BLA impaired consolidation of fear amygdala and the CA1 of rats given saline or a-FMHis infusions memories (18), whereas H3 receptor agonists ameliorated the ex- in the lateral ventricle (LV) 24 h before training. Controls re- pression of adverse memories (19). This effect involved H2 receptors ceived saline into the LV and no foot shock (untrained). pCREB and was accompanied by a bimodal modulation of the local cholin- levels were measured also in rats that received saline into the LV ergic tone (18, 19). and were placed directly over the electrified grid (saline/foot- Although these findings indicate that administration of hista- shocked) to control for potential changes in CREB phosphory- minergic ligands modulates memory consolidation, studies have lation due to the foot shock itself. Representative immunoblots not yet investigated the role of endogenous histamine in creating and the densitometric analysis are shown in Fig. 2 A (10 min) and memories for emotionally arousing training. To answer this ques- B (5 h). Ten min after training, pCREB levels measured in tion, we performed a first set of the experiments to examine how controlsandinsaline/foot-shocked animals were not signifi- brain histamine depletion obtained by using intralateral ventricle cantly different (Fig. 2A). Conversely, both amygdala and CA1 of (LV) administration of a-fluoromethylhistidine (a-FMHis), a sui- rats given saline or a-FMHis displayed a significant increase of cide inhibitor of HDC (20), affected IA memory processes. With pCREB density [one-way ANOVA and Bonferroni’smultiple the second set of experiments, we investigated IA training-induced comparison test (MCT), amygdala: F3,15 = 11.64; P < 0.0007; CA1: CREB phosphorylation in the brain of normal and histamine- F3,15 = 7.3; P < 0.004] compared with controls (Fig. 2A). Five hours depleted rats. The last set of experiments learned whether the local after training, no difference of pCREB density was found among infusion of histamine in the BLA or the CA1 region, respectively, the amygdala of controls and trained rats treated with saline or overcame a-FMHis–induced amnesia for IA training. a-FMHis (Fig. 2B, Left). In the CA1, however, only trained rats treated with saline, but not those with a-FMHis, showed a signifi- Results cant increase of pCREB density compared with controls (one-way Depletion of Histamine Impairs Long-Term but Not Short-Term ANOVA and Bonferroni’sMCT,F2,19 = 7.498; P < 0.004; Fig. 2B). Memory. Administration of a-FMHis (5 μg/μL) quickly sup- Therefore, a-FMHis administration caused amnesia of IA training pressed baseline and histamine H3 receptor antagonist-evoked on the 24-h retention test (Fig. 1B) and blocked the increase of release of histamine from the TMN of freely moving rats, as hippocampal pCREB associated with IA training (Fig. 2B). 180 min after injection, histamine release values decreased below the sensitivity of the method (Fig. S1). Histamine release was Effects of Histamine or 8-Bromoadenosine-3′,5′-cAMP Infusion into restored to control levels after approximately 3 d. the BLA on a-FMHis–Induced Amnesia. To further test the idea that To investigate the role of histamine in short- and long-term IA memory consolidation may depend on histaminergic signaling, memory retention, we examined the performance of rats treated we investigated whether administration of histamine (1 μg/μL) into with a-FMHis 24 h before training in the one-trial step-down the BLA counteracts the amnesic effect of a-FMHis. We tested also IA. The control group received intra-LV infusion of equivalent the membrane-permeable analog of cAMP, 8-bromoadenosine volume of saline. Retention test was carried out in different 3′,5′-cAMP (8Br-cAMP), considering that IA memory formation groups of animals at 2 h, 24 h, or 7 d after training. There were involved cAMP/cAMP-dependent protein kinase signaling pathway no differences in training performances in any group examined (22), and required CREB phosphorylation (24, 25). Rats received a- (Fig. 1). Latency of the a-FMHis group did not significantly FMHis or saline into LV 24 h before training, and saline, histamine, differ from that of controls at the 2-h retention test (Fig. 1A), but or 8Br-cAMP (1.25 μg/μL) into the BLA bilaterally immediately or it was significantly shorter at the 24-h retention test (unpaired 110 min after training. Retention test was carried out 24 h after t test, P < 0.0001 vs. controls; Fig. 1B). However, the latency of the training. Latency for all groups during training did not differ (Fig. 3 a-FMHis group and controls did not differ at the 7-d retention A and B). Fig. 3A shows the latency of rats given infusions of his- test (Fig. 1C), indicating that all animals formed a memory trace tamine or 8Br-cAMP into the BLA immediately after training. One- of the training experience. Restoration of histamine release to way ANOVA performed on the retention test revealed a significant F = P < control levels about 3 d after treatment with a-FMHis may ex- difference across groups ( 5,74 20.56; 0.0001). Further anal- plain this finding. To test this hypothesis, a-FMHis was admin- ysis with Bonferroni’sMCT(Fig.3A) showed that latencies of rats istered repeatedly on the second and sixth day after training. treated with a-FMHis (LV) and saline (BLA) were significantly Under this condition, memory retention tested 7 d after training shorter than those of all of the other groups, including animals was impaired (unpaired t test, P < 0.0001 vs. controls; Fig. 1D). treated with a-FMHis intra-LV and histamine or 8Br-cAMP intra- Thus, histamine depletion blocked long-term memory while BLA (Fig. 3A). Thus, both histamine and 8Br-cAMP fully antago- leaving short-term memory intact. The finding that a-FMHis had nized the amnesic effect of a-FMHis. Interestingly, infusion of ei- no effect on short-term memory rules out the possibility of its ther histamine or 8Br-cAMP per se enhanced step-down latency of influence on acquisition or retrieval mechanisms. Moreover, no rats treated with saline or with a-FMHis, compared with control changes in exploratory locomotor activity in the open field test group (Fig. 3A), thus suggesting a memory improving effect. Con- were observed between controls and a-FMHis–treated rats. versely, intra-BLA infusions of either histamine or 8Br-cAMP 110 min after training did not reverse a-FMHis–elicited amnesia Effect of Histamine Depletion on IA-Induced Increase of Cyclic Adenosine (Fig. 3B). Indeed, although one-way ANOVA performed on the Monophosphate Responsive-Element-Binding Protein Phosphorylation retention latency displayed a significant difference across groups in Rat BLA and Hippocampus. Cyclic adenosine monophosphate (F5,58 = 5.748; P < 0.0003), Bonferroni’s MCT analysis revealed (cAMP) responsive-element-binding protein (CREB) is a crucial that latencies of all rat groups infused with a-FMHis (LV) did not

2of7 | www.pnas.org/cgi/doi/10.1073/pnas.1506109112 Benetti et al. Downloaded by guest on October 1, 2021 differ significantly, and were significantly shorter compared with PNAS PLUS controls (Fig. 3B).

Effects of Histamine or 8Br-cAMP Infusion into Hippocampal CA1 Region on a-FMHis–Induced Amnesia. Rats received a-FMHis or sa- line into the LV 24 h before training, and saline, histamine, (1 μg/μl) or 8Br-cAMP (1.25 μg/μL) bilaterally into the CA1 at different posttraining times (Fig. 4, Top). Retention test was carried out 24 h after training. Latencies for all groups during training did not differ (Fig. 4 A–C). Histamine or 8Br-cAMP given immediately after training fully antagonized the effect of a-FMHis (one-way ANOVA and Bonferroni’sMCT,F5.63 = 31.12, P < 0.0001; Fig. 4A). Hista- mine or 8Br-cAMP fully antagonized the amnesic effect of a- FMHisalsowhenadministeredintoCA1110min(F5,62 = 6.762, P < 0.0001; Fig. 4B) or 6 h (F5,80 = 26.17, P < 0.0001; Fig. 4C) after training. Intra-CA1 infusion of histamine 12 h after training failed to antagonize a-FMHis–elicited amnesia, whereas 8Br- cAMP retained its effect (F5,74 = 23.5, P < 0.0001; Fig. 4D). Discussion Aversive memories can follow different processing routes, en- gaging multiple independent circuits. Emotionally relevant expe- riences activate the histaminergic system (26, 27), and there is an abundant literature demonstrating that activation of histamine receptors (mostly H2) in the BLA (18, 19), the dorsal hippocam- pus (15, 16) or the nucleus basalis magnocellularis (28) modulates the consolidation of memory associated to aversive events. Here we examined IA memory formation in rats temporarily NEUROSCIENCE depleted of histamine by LV injections of a-FMHis, an irre- versible histidine decarboxylase inhibitor, that completely sup- pressed spontaneous and evoked histamine release from the tuberomamillary nucleus. Our results provide strong evidence that intact histamine neurotransmission is required specifically for the establishment of long-term aversive memory, whereas short-term memory formation is independent of histamine neu- rotransmission. In fact, fear memories associated to IA were quickly forgotten when the brain histaminergic system was si- lenced. Furthermore, we found that memory can be restored in brain histamine-depleted rats, by local injections of histamine in the CA1 region of the dorsal hippocampus or the BLA, with a completely different time course: Whereas in the BLA only immediate posttraining infusion of histamine allows long-term memory formation, in the CA1, reinstatement of histamine consolidates aversive memory even 6 h after training, confirming that the hippocampus is engaged in IA memory processing for a period longer than the amygdala (2, 29). The unique finding in this study is that the histaminergic transmission in the BLA is crucial for the early phase of IA memory consolidation that occurred despite the blockade of hista- minergic neurotransmission in the hippocampus. This is surprising, given the important contribution of hippocampal histamine receptors to IA consolidation (15, 16). The different effects of histamine depletion on short- and long- term memories are in line with reports of several drug treatments impairing long-term memory, while leaving short-term memory intact (30–32). These findings suggest that short-term and long- term memories are separate processes, a view convincingly supported by pharmacological manipulations blocking short- term memory while keeping long-term memory intact, such as

Fig. 1. Effect of histamine acute depletion through a-FMHis administra- tion on IA task. The schematic drawings above A and D show the sequence nificantly shorter on the 24-h retention test. Data are expressed as means ± of procedures and treatment administrations. Rats implanted with an in- SEM of 8–15 animals for each group; unpaired t test, ****P < 0.0001 vs. fusion cannula in the LV received a-FMHis or saline (SAL) 24 h before controls. (D) a-FMHis or SAL was infused into the LV 24 h before and 2 and training. Retention test was performed 2 h (A), 24 h (B), and 7 d (C)after 6 d after training. Latencies of a-FMHis group were significantly shorter on training. Latencies of a-FMHis groups did not significantly differ from re- the 7-d retention test. Data are expressed as means ± SEM of 12–15 animals spective controls on the 2-h and the 7-d retention test, but they were sig- for each group (unpaired t test, ****P < 0.0001 vs. controls).

Benetti et al. PNAS Early Edition | 3of7 Downloaded by guest on October 1, 2021 Fig. 2. Effect of histamine (HA) depletion on the IA training-induced increase of CREB phosphorylation in rat amygdala and CA1. The schematic drawing on top of the figure displays the sequence of procedures and treatment administrations. Rats were implanted with an infusion cannula in the LV. (A) Repre- sentative immunoblots and densitometric quantification show that at 10 min after training pCREB/CREB ratio was increased in the amygdala and the CA1 of all rats undergone IA training, independently of having received saline or a-FMHis (n = 3–5 animals for each group). (B) At 5 h after training, pCREB/CREB ratio increased only in the CA1 of rats infused with SAL. Data are expressed as means ± SEM of 3–7 animals for each group; *P < 0.05, **P < 0.01 vs. controls, one- way ANOVA and Bonferroni’s MCT.

intraentorhinal cortex administration of AMPA receptor antagonist phosphorylation represents a crucial step for the establishment 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), GABAA receptor of long-term memories (21, 23, 25, 33), but is not a requirement agonist muscimol, or 5-HT1A receptor antagonist 1-(2-methoxy- for short-term memory formation in, e.g., conditioned taste phenyl)-4-(4-(2-phthalimido) butylpiperazine (NAN) (32). How- aversion (34). The present study reports that rats given either ever, when administered into the CA1 region, CNQX or muscimol saline or a-FMHis and submitted to the IA learning paradigm impair both short-term and long-term memory (32), thus sug- showed an increase of hippocampal and amygdalar CREB gesting selective involvement of different receptors in different phosphorylation 10 min after training, which is agreement with brain regions. previous reports (22, 33). The lack of difference between hista- Memory consolidation lasts several hours or days and relies on mine-depleted and not-depleted animals indicates that histamine molecular events occurring with different timings in several brain modulates long-term memory formation, influencing mechanisms regions, including the hippocampus and the amygdala (4). CREB other than early CREB activation in these two brain regions. Our

4of7 | www.pnas.org/cgi/doi/10.1073/pnas.1506109112 Benetti et al. Downloaded by guest on October 1, 2021 However, we did not observe an increase of pCREB in the PNAS PLUS amygdala 5 h after training, consistent with an active in- volvement of this structure only in the early phases of IA con- solidation (9, 35). A brief temporal window is supported also by experiments showing that reinstatement of histaminergic trans- mission in the BLA of histamine-depleted rats with exogenous histamine not only restored, but even improved the memory for IA, when local infusions of histamine were performed immedi- ately but not 110 min after training. The IA training activates molecular changes with different temporal progression in multiple brain areas, such as amygdala, hippocampus, entorhinal cortex, and parietal cortex (36, 37). However, experimental evidence indicates that CA1 and BLA operate also in parallel (9). Therefore, it is plausible that IA memory is spared after inactivation of the histaminergic system in the hippocampus because BLA histaminergic system takes over the process of consolidation, for example, enabling the of such information in other brain systems such as the entorhinal cortex or parietal cortex. Many reports have shown that IA training with high-intensity foot shocks reduced the memory impairment caused by hippocampal inactivation (38– 40), thus suggesting that consolidation of intense emotional ex- periences do not require a functioning hippocampus. However, this fear memory is acquired slowly and forgotten when remote memory is tested (41). We suggest that histamine in the BLA promoted inhibitory avoidance learning independently of hip- pocampus by increasing the emotional value of IA training. This NEUROSCIENCE effect is probably achieved through activation of histamine H2 receptors, whose responses occur through adenylyl cyclase stimula- tion (42). The activation of cAMP/PKA pathway, which targets CREB signaling, is crucial in IA memory processing (43). Moreover, many reports indicate that activation of H2 receptors potentiates the memory in aversive tasks, including IA (15, 16, 28). Consistently with this hypothesis, we showed that the nonhydrolysable cAMP analog, 8-Br-cAMP, produces effects similar to those of histamine. When given into the BLA immediately after training, both compounds rescued memory in histamine-depleted animals, but failed to produce this effect when they were administered 110 min after training. Thus, the involvement of BLA sufficient to exert IA long-term memory for- mation is required only for a brief temporal window. Also, when given in the CA1 of histamine-depleted rats, histamine and 8Br- cAMP had a similar effect in rescuing IA memory. However, hista- Fig. 3. Effect of HA or 8Br-cAMP infusion into the BLA on the amnesia of IA mine was effective up to 6 h after training, whereas 8Br-cAMP res- training induced by a-FMHis. The schematic drawing on top of the figure displays the sequence of procedures and treatment administrations. Rats cued the memory of IA training in histamine-depleted rats also when were implanted with infusion cannulae in the LV and in the BLA bilaterally. given 12 h after training. Molecular changes in the hippocampus of Rats receiving saline into both the LV and BLA served as controls. Retention rats trained with IA begin immediately after training and progress for test was performed 24 h after training. (A) HA, 8Br-cAMP, or SAL was bi- approximately 20 h (36, 44). The effect of histamine lasts up to 6 h; laterally infused into the BLA immediately after training. Data are expressed however, the involvement of other hippocampal modulatory neuro- ± – < < as means SEM of 9 15 animals for each group; *P 0.05, ***P 0.001 vs. transmission that might contribute to a later phase of memory con- controls, ####P < 0.0001 vs. “a-FMHis (LV) + HA (BLA)” and “a-FMHis (LV) + ” ’ solidation cannot be ruled out (22). 8Br-cAMP (BLA), one-way ANOVA, and Bonferroni s MCT. (B) HA, 8Br- i cAMP, or SAL was bilaterally infused into the BLA 110 min after training. Taken together, the present findings suggest that ( ) the in- Data are expressed as means ± SEM of 8–24 animals for each group; **P < tegrity of the histaminergic system in the brain is crucial for IA 0.01 vs. controls, one-way ANOVA and Bonferroni’s MCT. long-term, but not short-term, memory formation; (ii) histamine- depleted rats did not express long-term aversive memory and dis- played a lack of hippocampal CREB phosphorylation; (iii)long- results suggest instead that histamine activates the CREB pathway term memory was formed immediately following posttraining to exert its effects later during the temporal progression restoration of histamine transmission in the BLA independently of memory consolidation. Indeed, pCREB levels were augmented 5 of hippocampus. Therefore, despite current views conferring to the h after training only in the CA1 of saline-treated rats that well re- BLA essentially a modulatory function in IA memory formation, membered the IA training. This event did not occur in the CA1 of this and other evidence (9) indicates that it plays a role also in a-FMHis–treated rats, which displayed long-term memory impair- consolidation of this task. A prudent interpretation of these results ments. This observation fits well with previous reports that IA is that following the local activation of the histaminergic system, training increases hippocampal pCREB levels 3–6 h after training the BLA takes over the functions of the hippocampus in the (22), suggesting a causal correlation between pCREB levels in the consolidation process and renders hippocampus no longer crucial hippocampus and long-term memory formation (23, 33). for long-term memory formation.

Benetti et al. PNAS Early Edition | 5of7 Downloaded by guest on October 1, 2021 Fig. 4. Effect of HA or 8Br-cAMP infusion into the CA1 region of hippocampus on the amnesia of IA training induced by a-FMHis. The schematic drawing on top of the figure displays the sequence of procedures and treatment administrations. Rats were implanted with infusion cannulae in the LV and in the CA1 bilaterally. Rats receiving saline into both the LV and CA1 served as controls. Retention test was carried out 24 h after training. HA, 8Br-cAMP, or SAL were bilaterally infused into the CA1. (A) Immediately after training. Data are expressed as means ± SEM of 10–12 animals for each group; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. controls, ####P < 0.0001 vs. “a-FMHis (LV) + HA (CA1),” $$$$P < 0.0001 vs. “a-FMHis (LV) + 8Br-cAMP (CA1).” (B) One hundred ten minutes after training. Data are expressed as means ± SEM of 7–19 animals for each group; ***P < 0.001 vs. controls, ###P < 0.001 vs. “a-FMHis (LV) + HA (CA1),” $P < 0.05 vs. “a-FMHis (LV) + 8Br-cAMP (CA1).” (C) Six hours after training. Data are expressed as means ± SEM of 11–17 animals for each group; ****P < 0.0001 vs. controls, ####P < 0.0001 vs. “a-FMHis (LV) + HA (CA1),” $$$$P < 0.0001 vs. “a-FMHis (LV) + 8Br-cAMP (CA1).” (D) Twelve hours after training. Data are expressed as means ± SEM of 11–17 animals for each group; **P < 0.01, ****P < 0.0001 vs. controls, $$$$P < 0.0001 vs. “a-FMHis (LV) + 8Br-cAMP (CA1)” (one-way ANOVA and Bonferroni’sMCT).

Materials and Methods allowed 7 d to recover from surgery before behavioral procedures. Animals were Animals. Male Wistar rats (3 mo old, 300–330 g) purchased from Centro de handled once daily for three consecutive days, and all behavioral procedures was Reprodução e Experimentação de Animais de Laboratorio of the Uni- conducted between 8:00 and 11:00 AM. versidade Federal do Rio Grande do Sul (our regular provider) were used. × × They were housed four to a cage with water and food ad libitum, under a Inhibitory Avoidance Task. The apparatus consisted in a 50 25 25 cm 12-h light/dark cycle (lights on at 7:00 AM). The temperature of the animal Plexiglas box with a 5-cm-high, 8-cm-wide, 25-cm-long Formica platform on room was maintained at 22–24 °C. All procedures were in accordance with the left end of a grid of 1-mm caliber bronze bars spaced 0.8 mm apart. The the National Institutes of Health’s Guide for the Care and Use of Laboratory rats were gently placed on the platform facing the left rear corner. When Animals (45) and were approved by Pontifical Catholic University of Rio they stepped down, placing their four paws on the grid, they received a 2-s Grande do Sul. 0.5-mA scrambled foot shock and then were immediately withdrawn from the training box. Retention test was carried out 2 h, 24 h, or 7 d after the Surgery. At least 1 wk after their arrival, the animals were anesthetized training session. The procedure was the same except that the foot shock was (75 mg/kg ketamine plus 10 mg/kg xylazine) and placed on a stereotaxic frame omitted. In the retention test, the step-down latency was 300 s. Latency to (Stellar; Stoeling). A stainless steel cannula (22 gauge) was implanted in the step down was measured with an automated stopwatch. LV and fixed to the skull by using dental cement, according to the following coordinates: anterior, −0.9 mm; lateral, −1.5 mm; ventral, −3.6 mm (46), and Drugs and Infusion Procedures. At the time of drug microinfusions, the ani- used for a-FMHis administration. Rats were also implanted bilaterally with mals were gently restrained by hand, and the injection needle (30 gauge) was 22-gauge guide cannulae 1 mm above the CA1 area of the hippocampus or fitted tightly into the guides, extending 1 mm from the tip of the guide 1 mm above the BLA. The coordinates were anterior, −4.2 mm; lateral, cannulae. The injection needle was connected to a 10-μL Hamilton micro- ±3.0 mm; ventral, −1.8 mm for the CA1, and anterior, −2.4 mm; lateral, ±5.1 mm; syringe, and the infusions were performed at a rate of 0.5 μL/30 s. The in- ventral, −7.5 mm for the BLA (46). Cannulae placements were verified post- fusion cannula was left in place for an additional 60 s to minimize backflow. mortem as described in detail in SI Materials and Methods.Animalswere It was then carefully withdrawn and placed on the other side, where the

6of7 | www.pnas.org/cgi/doi/10.1073/pnas.1506109112 Benetti et al. Downloaded by guest on October 1, 2021 procedure was repeated. The entire bilateral infusion procedure took ap- phosphatase inhibitors [50 mM Tris·HCl (pH 7.5), 50 mM NaCl, 10 mM EGTA, PNAS PLUS proximately 90 s. Infusions into the BLA were performed immediately or 5 mM EDTA, 2 mM sodium pyrophosphate, 4 mM p-nitrophenyl phosphate, 110 min after training; into the CA1 immediately; or 110 min, 3 h or 6 h after 1mMNa3VO4, 1.1 mM PMSF, 20 μg/μL leupeptin, 50 μg/μL aprotinin, 0.1% μ μ training. The drugs used were histamine (1 g/uL) and 8Br-cAMP (1.25 g/uL) SDS] and centrifuged at 13.8 × g at 4 °C for 15 min. The following procedure μ purchased from Sigma-Aldrich. a-FMHis (5 g/uL) was synthesized at Abbott is described in detail in SI Materials and Methods. For each sample, a ratio of Laboratories. The doses were chosen among those found to be effective in pSer133-CREB/CREB densities was calculated, and then all of the individual previous papers from our group or others (6, 7). The drugs had no effects on rates were expressed as a percentage of the average of ratios obtained from locomotion, exploration, or performance in an elevated plus maze (6). The volume of drugs infused was 0.5 μL per side in the BLA and 1 μL per side into control group. the CA1 area and LV. Control groups received equal volumes of sterile saline (0.9%). Data and Statistical Analysis. Statistical analysis was performed by using Prism Software (GraphPad). Data are expressed as means ± SEM. Inhibitory avoidance Microdialysis Experiments. The effects of a-FMHis on brain histamine release latencies as well as pCREB/CREB ratio were analyzed with unpaired t test or were evaluated in freely moving rats implanted with microdialysis probes one-way ANOVA. The source of the detected significances was determined by (Fig. S1). For details on surgery, experimental protocols, and HPLC-fluori- Bonferroni’s multiple comparison post hoc test. P values less than 0.05 were metric assay to quantify histamine, see SI Materials and Methods. considered statistically significant. The number of rats per group is indicated in the figure legends. Western Blotting Analysis. Animals were killed 10 min or 5 h after training, the brain was dissected out on ice, and the amygdala and the CA1 were im- ACKNOWLEDGMENTS. We thank Dr. M. Cowart for the synthesis of α-FMHis. mediately isolated. The pooled structures (left and right) were individually This work was supported by CNPq Grant 400289/2012-1 (Brazil), Compagnia homogenized in 200 μL of ice-cold lysis buffer containing protease and di San Paolo (Italy), and Ente Cassa di Risparmio di Firenze (Italy).

1. McGaugh JL (2013) Making lasting memories: Remembering the significant. Proc Natl avoidance learning: Abolition by NMDA receptor blockade. Brain Res Mol Brain Res Acad Sci USA 110(Suppl 2):10402–10407. 76(1):36–46. 2. Izquierdo I, Medina JH (1997) Memory formation: The sequence of biochemical events 26. Westerink BH, et al. (2002) Evidence for activation of histamine H3 autoreceptors in the hippocampus and its connection to activity in other brain structures. Neurobiol during handling stress in the prefrontal cortex of the rat. Synapse 43(4):238–243. Learn Mem 68(3):285–316. 27. Valdés JL, et al. (2010) The histaminergic tuberomammillary nucleus is critical for 3. Euston DR, Gruber AJ, McNaughton BL (2012) The role of medial prefrontal cortex in motivated arousal. Eur J Neurosci 31(11):2073–2085. memory and decision making. Neuron 76(6):1057–1070. 28. Benetti F, Baldi E, Bucherelli C, Blandina P, Passani MB (2013) Histaminergic ligands 4. Izquierdo I, et al. (2006) Different molecular cascades in different sites of the brain injected into the nucleus basalis magnocellularis differentially affect fear condition- – – control memory consolidation. Trends Neurosci 29(9):496 505. ing consolidation. Int J Neuropsychopharmacol 16(3):575 582. NEUROSCIENCE 5. Phelps EA (2004) Human : Interactions of the amygdala and 29. Katche C, Cammarota M, Medina JH (2013) Molecular signatures and mechanisms of hippocampal complex. Curr Opin Neurobiol 14(2):198–202. long-lasting memory consolidation and storage. Neurobiol Learn Mem 106:40–47. 6. Kim JJ, Fanselow MS (1992) Modality-specific of fear. Science 30. Yin JCP, Tully T (1996) CREB and the formation of long-term memory. Curr Opin 256(5057):675–677. Neurobiol 6(2):264–268. 7. Zelikowsky M, et al. (2013) Prefrontal microcircuit underlies contextual learning after 31. Izquierdo LA, et al. (2000) Short- and long-term memory are differentially affected by hippocampal loss. Proc Natl Acad Sci USA 110(24):9938–9943. metabolic inhibitors given into hippocampus and entorhinal cortex. Neurobiol Learn 8. Valtonen J, Gregory E, Landau B, McCloskey M (2014) New learning of music after Mem 73(2):141–149. bilateral medial temporal lobe damage: Evidence from an amnesic patient. Front 32. Izquierdo I, et al. (1998) Mechanisms for memory types differ. Nature 393(6686): Hum Neurosci 8:694. 635–636. 9. Cammarota M, et al. (2008) Parallel memory processing by the CA1 region of the 33. Morris KA, Gold PE (2012) Age-related impairments in memory and in CREB and dorsal hippocampus and the basolateral amygdala. Proc Natl Acad Sci USA 105(30): pCREB expression in hippocampus and amygdala following inhibitory avoidance 10279–10284. training. Mech Ageing Dev 133(5):291–299. 10. Roozendaal B, McGaugh JL (2011) Memory modulation. Behav Neurosci 125(6): 34. Lamprecht R, Hazvi S, Dudai Y (1997) cAMP response element-binding protein in the 797–824. amygdala is required for long- but not short-term conditioned taste aversion mem- 11. de Almeida MA, Izquierdo I (1986) Memory facilitation by histamine. Arch Int Phar- ory. J Neurosci 17(21):8443–8450. macodyn Ther 283(2):193–198. 35. Bevilaqua L, et al. (1997) Drugs acting upon the cyclic adenosine monophosphate/ 12. Haas HL, Sergeeva OA, Selbach O (2008) Histamine in the nervous system. Physiol Rev protein kinase A signalling pathway modulate memory consolidation when given late 88(3):1183–1241. after training into rat hippocampus but not amygdala. Behav Pharmacol 8(4): 13. Panula P, Pirvola U, Auvinen S, Airaksinen MS (1989) Histamine-immunoreactive 331–338. nerve fibers in the rat brain. Neuroscience 28(3):585–610. 36. Bambah-Mukku D, Travaglia A, Chen DY, Pollonini G, Alberini CM (2014) A positive 14. Panula P, Nuutinen S (2013) The histaminergic network in the brain: Basic organi- autoregulatory BDNF feedback loop via C/EBPβ mediates hippocampal memory con- zation and role in disease. Nat Rev Neurosci 14(7):472–487. solidation. J Neurosci 34(37):12547–12559. 15. da Silva WC, Bonini JS, Bevilaqua LR, Izquierdo I, Cammarota M (2006) Histamine 37. Izquierdo I, et al. (1997) Sequential role of hippocampus and amygdala, entorhinal enhances inhibitory avoidance memory consolidation through a H2 receptor-de- cortex and parietal cortex in formation and retrieval of memory for inhibitory pendent mechanism. Neurobiol Learn Mem 86(1):100–106. avoidance in rats. Eur J Neurosci 9(4):786–793. 16. Giovannini MG, et al. (2003) Improvement in fear memory by histamine-elicited ERK2 38. Prada-Alcala RA, Medina AC, Lopez NS, Quirarte GL (2012) Intense emotional expe- activation in hippocampal CA3 cells. J Neurosci 23(27):9016–9023. riences and enhanced training prevent memory loss induced by post-training amnesic 17. Adams JP, Sweatt JD (2002) Molecular psychology: Roles for the ERK MAP kinase treatments administered to the striatum, amygdala, hippocampus or substantia ni- cascade in memory. Annu Rev Pharmacol Toxicol 42:135–163. gra. Rev Neurosci 23(5-6):501–508. 18. Passani MB, et al. (2001) Histamine H3 receptor-mediated impairment of contextual 39. Quiroz C, et al. (2003) Enhanced inhibitory avoidance learning prevents the memory- fear conditioning and in-vivo inhibition of cholinergic transmission in the rat baso- impairing effects of post-training hippocampal inactivation. Exp Brain Res 153(3): lateral amygdala. Eur J Neurosci 14(9):1522–1532. 400–402. 19. Cangioli I, et al. (2002) Activation of histaminergic H3 receptors in the rat basolateral 40. Garín-Aguilar ME, Medina AC, Quirarte GL, McGaugh JL, Prado-Alcalá RA (2014) In- amygdala improves expression of fear memory and enhances release. tense aversive training protects memory from the amnestic effects of hippocampal Eur J Neurosci 16(3):521–528. inactivation. Hippocampus 24(1):102–112. 20. Garbarg M, Barbin G, Rodergas E, Schwartz JC (1980) Inhibition of histamine synthesis 41. Poulos AM, et al. (2009) Persistence of fear memory across time requires the baso- in brain by alpha-fluoromethylhistidine, a new irreversible inhibitor: in Vitro and in lateral amygdala complex. Proc Natl Acad Sci USA 106(28):11737–11741.

vivo studies. J Neurochem 35(5):1045–1052. 42. Baudry M, Martres MP, Schwartz J-C (1975) H1 and H2 receptors in the histamine- 21. Carlezon WAJ, Jr, Duman RS, Nestler EJ (2005) The many faces of CREB. Trends induced accumulation of cyclic AMP in guinea pig brain slices. Nature 253(5490): Neurosci 28(8):436–445. 362–364. 22. Bernabeu R, et al. (1997) Involvement of hippocampal cAMP/cAMP-dependent pro- 43. Bernabeu R, Schmitz P, Faillace MP, Izquierdo I, Medina JH (1996) Hippocampal cGMP tein kinase signaling pathways in a late memory consolidation phase of aversively and cAMP are differentially involved in memory processing of inhibitory avoidance motivated learning in rats. Proc Natl Acad Sci USA 94(13):7041–7046. learning. Neuroreport 7(2):585–588. 23. Viola H, et al. (2000) Phosphorylated cAMP response element-binding protein as a 44. Taubenfeld SM, Milekic MH, Monti B, Alberini CM (2001) The consolidation of new molecular marker of memory processing in rat hippocampus: Effect of novelty. but not reactivated memory requires hippocampal C/EBPbeta. Nat Neurosci 4(8): J Neurosci 20(23):RC112. 813–818. 24. McGaugh JL, Izquierdo I (2000) The contribution of pharmacology to research on the 45. Committee on Care and Use of Laboratory Animals (1985) Guide for the Care and Use mechanisms of memory formation. Trends Pharmacol Sci 21(6):208–210. of Laboratory Animals (Natl Inst Health, Bethesda). 25. Cammarota M, et al. (2000) Learning-associated activation of nuclear MAPK, CREB 46. Paxinos G, Watson C (1998) The Rat Brain in Stereotaxic Coordinates (Academic, and Elk-1, along with Fos production, in the rat hippocampus after a one-trial New York).

Benetti et al. PNAS Early Edition | 7of7 Downloaded by guest on October 1, 2021