Skip to main content

Optogenetic inactivation of the medial septum impairs long-term object recognition memory formation

Abstract

Theta is one of the most prominent extracellular synchronous oscillations in the mammalian brain. Hippocampal theta relies on an intact medial septum (MS) and has been consistently recorded during the training phase of some learning paradigms, suggesting that it may be implicated in hippocampus-dependent long-term memory processing. Object recognition memory (ORM) allows animals to identify familiar items and is essential for remembering facts and events. In rodents, long-term ORM formation requires a functional hippocampus but the involvement of the MS in this process remains controversial. We found that training adult male Wistar rats in a long-term ORM-inducing learning task involving exposure to two different, but behaviorally equivalent novel stimuli objects increased hippocampal theta power, and that suppressing theta via optogenetic MS inactivation caused amnesia. Importantly, the amnesia was specific to the object the animals were exploring when the MS was inactivated. Taken together, our results indicate that the MS is necessary for long-term ORM formation and suggest that hippocampal theta activity is causally linked to this process.

Main text

Neural oscillations are repetitive rhythmic patterns of electrical activity that occur spontaneously or in response to stimuli. Theta is a slow (5–10 Hz) neural oscillation predominantly found in the hippocampus, particularly in the CA1 region, where it is more regular and shows maximum amplitude [1]. Hippocampal theta is sensitive to medial septum (MS) lesions [2] and, although its behavioral correlates have not yet been fully elucidated, extensive evidence indicates that it supports learning [3,4,5]. Indeed, theta facilitates hippocampal long-term potentiation (LTP) [6], the main cellular model of hippocampus-dependent long-term memory. Object recognition memory (ORM) allows animals to determine the familiarity of items and is vital for remembering events and planning actions. In rodents, training in an ORM-based learning paradigm activates several plasticity-related signaling pathways and induces LTP in dorsal CA1, indicating that the hippocampus is essential for long-term ORM formation [7,8,9]. Conversely, the participation of the MS in this process remains controversial. For example, septal lesions that impair spatial and working memory do not affect long-term ORM [10, 11] but MS stimulation attenuates the long-term ORM deficit observed in epileptic mice by increasing hippocampal theta activity [12]. Therefore, we set out to analyze whether MS-regulated hippocampal theta is indeed associated with long-term ORM retention. Firstly, we determined whether long-term ORM formation affects hippocampal theta. To do that, we implanted electrode arrays in the dorsal CA1 region of adult male Wistar rats (3-months-old, 300–350 g) and trained them in the novel object-recognition paradigm, a long-term ORM-inducing task based on the rodents’ natural predilection for novelty that involves exposure to two different but behaviorally equivalent novel objects A and B in a familiar open field arena for 5-min (Fig. 1a) [13]. A digital video camera fixed above the arena was utilized for tracking, recording, and analyzing the animals’ position and behavior with the ObjectScan system software (for details see Additional file 1). Exploration events were defined as the ≥ 0.5-s-long epochs during which the animals sniffed and/or touched the stimuli objects with their muzzle and/or forepaws. All other epochs ≥ 0.5 s in duration were regarded as inter-exploration events and, of these, we further considered only those during which the mean locomotion speed was ≤ the mean locomotion speed of all exploration events. Events lasting < 0.5-s were excluded from analysis. Local field potentials (LFP) were recorded continuously during the training session. Signals were amplified, digitized, filtered at cutoff frequencies of 0.3 and 250 Hz, and sampled at 1 kHz. Data from time windows corresponding to exploration and inter-exploration events were extracted and analyzed offline using built-in or custom-written routines (see Additional file 1 for details). As expected, the exploration time and the number of exploration events during training did not differ between objects A and B (Fig. 1b; t (5) = 0.79, P = 0.46 for exploration time; t (5) = 1.21, P = 0.28 for exploration events in paired t test). Exploratory activity was observed all through the training session (Fig. 1b). Theta activity was also evident throughout this session (Fig. 1c), but theta power, which predicts learning [14], was particularly high during object exploration (Fig. 1d, e). Indeed, power spectra analysis showed that theta power during object exploration epochs was 36 ± 7% higher than during inter-exploration periods (Fig. 1f, g; F (2, 10) = 15.55; P = 0.0009. Obj A vs IE, P = 0.002, Obj B vs IE, P = 0.001 in Bonferroni’s multiple comparisons test after RM one-way ANOVA). Theta peak frequency did not differ between exploration and inter-exploration events (Fig. 1f; F (2, 10) = 3.29; P = 0.079 in RM one-way ANOVA). Neither the power nor the peak frequency of theta differed between object A and object B exploration epochs (Fig. 1f; Obj A vs Obj B, P > 0.99 for theta power; Obj A vs Obj B, P = 0.13 for peak frequency in Bonferroni’s multiple comparisons test after RM one-way ANOVA). One day after training, long-term ORM retention was evaluated by re-exposing animals to familiar object A and novel object C. As expected, the animals preferentially explored the novel object at test (TT; Fig. 1h; t (5) = 6.95, P = 0.0009 in one sample t test with theoretical mean = 50).

Fig. 1
figure 1

ah Hippocampal theta activity increases during training in the novel object recognition task. Male Wistar rats implanted with electrode arrays in the CA1 region of the dorsal hippocampus (n = 6) were habituated to an open-field arena (HAB; 20-min/d/4d) and 24-h later trained in the novel object-recognition task (TR). Long-term ORM was evaluated 24-h thereafter (TT). a (Top) Graphic representation of the experimental design. (Bottom) Behavioral data showing exploration events distribution and hippocampal LFP recordings during TR for a representative rat. b (Top) Mean exploration time (% and s), mean number of exploration events, and representative trajectory during TR. (Bottom) Mean exploration time and number of exploration events per minute during TR. c (Top) Exploration events and theta activity during TR for a representative rat. (Bottom) Representative power spectral density plots during TR. d (Top) Spectrograms highlighting theta activity during exploration and inter-exploration (IE) events. (Middle) Mean locomotion speed during exploration events and IE. (Bottom) Representative power spectral density plots for exploration events and IE. e (Top) Mean theta power for exploration events and IE computed for interquartile intervals. (Bottom) Representative plot showing cumulative theta power during TR. f (Left) Mean power spectral densities for exploration and IE during TR. (Top right) Mean theta power and (Bottom right) mean theta peak frequency during TR. g (Top) Raw data, filtered theta and theta envelope for a representative exploration event. (Bottom) Spatial distribution of theta during TR for a representative rat. h (Top) Discrimination index during TR and TT. (Bottom) Mean exploration time (%) during TT. in Optogenetic inactivation of the medial septum causes amnesia. Two groups of rats expressing Arch-T in MS (LightOFF, n = 22 and LightON A, n = 19) were trained in the novel object recognition task exactly as in A, except that LightON A animals received yellow light (565-nm) stimulation during object A exploration events whereas LightOFF animals were not stimulated. Long-term ORM was evaluated 24-h later. i Graphic representation of the experimental design. j Trajectory during TR for representative LightOFF and LightON A rats. k Mean distance travelled during TR and TT. l Mean exploration time (s) and number of exploration events during TR and TT. m Discrimination index during TR and TT. n Mean exploration time (%) during TR and TT. Individual values in Additional file 1: Tables S1, S2

Normal MS functioning is essential for hippocampal theta activity [2]. In fact, MS inactivation has been used before as a tool to abolish hippocampal theta during learning [15]. Previously, we showed that yellow light (565 nm) stimulation of the MS of rats expressing the yellow light-sensing optical neural silencer archaerhodopsin T (ArchT; see Additional file 1 for technical details) [16] rapidly and reversibly cancels theta in dorsal CA1 [17]. Therefore, to analyze the involvement of the MS in long-term ORM formation and to further assess whether hippocampal theta is indeed linked to this process, rats expressing ArchT in the MS were trained in the novel object-recognition paradigm using A and B as stimuli objects, and yellow light was delivered to the MS just during object A exploration (Fig. 1i). This procedure did not affect locomotor activity (Fig. 1j, k; t (39) = 1.29, P = 0.20 for LigthOFF vs LightON A in unpaired t test), object exploration time (Fig. 1l; t (39) = 1.33, P = 0.18 for LigthOFF vs LightON A in unpaired t test), or the number of exploration events (Fig. 1l; t (39) = 1.93, P = 0.06 for LigthOFF vs LightON A in unpaired t test). Long-term ORM was evaluated during a retention test session in the presence of familiar object A or familiar object B alongside novel object C carried out 24-h post-training. We found that unstimulated ArchT-expressing animals discriminated objects A and B from novel object C (Fig. 1m, n; t (10) = 5.96, P < 0.0001 for test AC, t (10) = 7.48, P < 0.0001 for test BC in one sample t test with theoretical mean = 50); however, rats that had been delivered yellow light on the MS during object A exploration at training discriminated object B but not object A from novel object C at test (Fig. 1m, n; t (9) = 1.38, P = 0.19 for test AC, t (8) = 7.30, P < 0.0001 for test BC in one sample t test with theoretical mean = 50).

Hippocampal theta amplitude depends on locomotion speed [18], but it is unlikely that changes in this variable could account for the increase in theta power that we observed during training because we only compared exploration events with inter-exploration events matched for similar speed. It is also unlikely that the amnesia triggered by MS inactivation was due to impaired recall, subpar training performance, optogenetic construct overexpression, or a harmful effect of light stimulation per se, because it was specific to the object the animals were exploring when optogenetic suppression was applied, and light delivery did not affect object exploration. The MS not only projects to the hippocampus but also to the anterior cingulate cortex (ACC) [19]. Therefore, the amnesia induced by MS inactivation could potentially be caused by impairment of this interaction. However, the ACC is not involved in long-term ORM formation [20] and inhibition of MS-ACC projections does not affect this form of declarative-like memory [21]. Hence, it is implausible that disruption of ACC function could account for our results which are likely due to hippocampal theta inhibition. The notion that the hippocampus is required for ORM processing has received wide experimental support, but it is not unanimously accepted [22]. For example, pre-training intra-hippocampal muscimol administration affects ORM only when the training-test interval is longer than 10 min [23], suggesting that the hippocampus is not required for short-term ORM recall, that other brain regions take over the role of the hippocampus in short-term ORM processing when it remains disabled for a long time, or that short-term and long-term ORM involve independent mechanisms, as it has been reported for other memory types [24]. In this regard, our data indicate that the hippocampus is key for long-term ORM formation and substantiate further the idea that the two long-term object memories acquired during training in the novel object recognition task are independent [13]. Furthermore, the fact that the animals were amnesic only for the object they were exploring when the MS was inactivated strongly indicates that theta is not just a byproduct of learning-induced neural plasticity but is functionally linked to the calculations that occur in the hippocampus during long-term ORM formation.

Availability of data and materials

Please contact the corresponding author for data requests.

Abbreviations

CA1:

Cornu Ammonis-1

LFP:

Local field potentials

LTP:

Long-term potentiation

ORM:

Object recognition memory

TT:

Test

References

  1. Buzsáki G. Theta oscillations in the hippocampus. Neuron. 2002;33(3):325–40. https://doi.org/10.1016/s0896-6273(02)00586-x.

    Article  PubMed  Google Scholar 

  2. Yoder RM, Pang KC. Involvement of GABAergic and cholinergic medial septal neurons in hippocampal theta rhythm. Hippocampus. 2005;15(3):381–92. https://doi.org/10.1002/hipo.20062.

    Article  CAS  PubMed  Google Scholar 

  3. Seager MA, Johnson LD, Chabot ES, Asaka Y, Berry SD. Oscillatory brain states and learning: Impact of hippocampal theta-contingent training. Proc Natl Acad Sci USA. 2002;99(3):1616–20. https://doi.org/10.1073/pnas.032662099 (Epub 2002 Jan 29).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Düzel E, Penny WD, Burgess N. Brain oscillations and memory. Curr Opin Neurobiol. 2010;20(2):143–9. https://doi.org/10.1016/j.conb.2010.01.004 (Epub 2010 Feb 22).

    Article  CAS  PubMed  Google Scholar 

  5. Berry SD, Seager MA. Hippocampal theta oscillations and classical conditioning. Neurobiol Learn Mem. 2001;76(3):298–313. https://doi.org/10.1006/nlme.2001.4025.

    Article  CAS  PubMed  Google Scholar 

  6. Huerta PT, Lisman JE. Synaptic plasticity during the cholinergic theta-frequency oscillation in vitro. Hippocampus. 1996;6(1):58–61. https://doi.org/10.1002/(SICI)10981063(1996)6:1%3c58:AID-HIPO10%3e3.0.CO;2-J.

    Article  CAS  PubMed  Google Scholar 

  7. Ill-Raga G, Köhler C, Radiske A, Lima RH, Rosen MD, Muñoz FJ, Cammarota M. Consolidation of object recognition memory requires HRI kinase-dependent phosphorylation of eIF2α in the hippocampus. Hippocampus. 2013;23(6):431–6. https://doi.org/10.1002/hipo.22113 (Epub 2013 Mar 18).

    Article  CAS  PubMed  Google Scholar 

  8. Myskiw JC, Rossato JI, Bevilaqua LR, Medina JH, Izquierdo I, Cammarota M. On the participation of mTOR in recognition memory. Neurobiol Learn Mem. 2008;89(3):338–51. https://doi.org/10.1016/j.nlm.2007.10.002 (Epub 2007 Nov 26).

    Article  CAS  PubMed  Google Scholar 

  9. Clarke JR, Cammarota M, Gruart A, Izquierdo I, Delgado-García JM. Plastic modifications induced by object recognition memory processing. Proc Natl Acad Sci USA. 2010;107(6):2652–7. https://doi.org/10.1073/pnas.0915059107 (Epub 2010 Jan 25).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Kornecook TJ, Kippin TE, Pinel JP. Basal forebrain damage and object-recognition in rats. Behav Brain Res. 1999;98(1):67–76. https://doi.org/10.1016/s0166-4328(98)00053-9.

    Article  CAS  PubMed  Google Scholar 

  11. Cai L, Gibbs RB, Johnson DA. Recognition of novel objects and their location in rats with selective cholinergic lesion of the medial septum. Neurosci Lett. 2012;506(2):261–5. https://doi.org/10.1016/j.neulet.2011.11.019 (Epub 2011 Nov 20).

    Article  CAS  PubMed  Google Scholar 

  12. Wang Y, Shen Y, Cai X, Yu J, Chen C, Tan B, Tan N, Cheng H, Fan X, Wu X, Liu J, Wang S, Wang Y, Chen Z. Deep brain stimulation in the medial septum attenuates temporal lobe epilepsy via entrainment of hippocampal theta rhythm. CNS Neurosci Ther. 2021;27(5):577–86. https://doi.org/10.1111/cns.13617 (Epub 2021 Jan 27).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gonzalez MC, Rossato JI, Radiske A, Bevilaqua LRM, Cammarota M. Dopamine controls whether new declarative information updates reactivated memories through reconsolidation. Proc Natl Acad Sci USA. 2021;118(29):e2025275118. https://doi.org/10.1073/pnas.2025275118.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Backus AR, Schoffelen JM, Szebényi S, Hanslmayr S, Doeller CF. Hippocampal prefrontal theta oscillations support memory integration. Curr Biol. 2016;26(4):450–7. https://doi.org/10.1016/j.cub.2015.12.048 (Epub 2016 Jan 28).

    Article  CAS  PubMed  Google Scholar 

  15. Gemzik ZM, Donahue MM, Griffin AL. Optogenetic suppression of the medial septum impairs working memory maintenance. Learn Mem. 2021;28(10):361370. https://doi.org/10.1101/lm.053348.120.

    Article  CAS  Google Scholar 

  16. Han X, Chow BY, Zhou H, Klapoetke NC, Chuong A, Rajimehr R, Yang A, Baratta MV, Winkle J, Desimone R, Boyden ES. A high-light sensitivity optical neural silencer: development and application to optogenetic control of non-human primate cortex. Front Syst Neurosci. 2011;5:18. https://doi.org/10.3389/fnsys.2011.00018.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Radiske A, Gonzalez MC, Conde-Ocazionez S, Rossato JI, Köhler CA, Cammarota M. Cross-frequency phase-amplitude coupling between hippocampal theta and gamma oscillations during recall destabilizes memory and renders it susceptible to reconsolidation disruption. J Neurosci. 2020;40(33):6398–408. https://doi.org/10.1523/JNEUROSCI.0259-20.2020 (Epub 2020 Jul 13).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. McFarland WL, Teitelbaum H, Hedges EK. Relationship between hippocampal theta activity and running speed in the rat. J Comp Physiol Psychol. 1975;88(1):324–8. https://doi.org/10.1037/h0076177.

    Article  CAS  PubMed  Google Scholar 

  19. Senut MC, Menetrey D, Lamour Y. Cholinergic and peptidergic projections from the medial septum and the nucleus of the diagonal band of Broca to dorsal hippocampus, cingulate cortex and olfactory bulb: a combined wheatgerm agglutinin-apohorseradish peroxidase-gold immunohistochemical study. Neuroscience. 1989;30(2):385–403. https://doi.org/10.1016/0306-4522(89)90260-1.

    Article  CAS  PubMed  Google Scholar 

  20. Pezze MA, Marshall HJ, Fone KC, Cassaday HJ. Role of the anterior cingulate cortex in the retrieval of novel object recognition memory after a long delay. Learn Mem. 2017;24(7):310–7. https://doi.org/10.1101/lm.044784.116.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Jin T, Chen R, Shao M, Yang X, Ma L, Wang F. Dorsal hippocampus- and ACC-projecting medial septum neurons differentially contribute to the recollection of episodic-like memory. FASEB J. 2020;34(9):11741–53. https://doi.org/10.1096/fj.202000398R (Epub 2020 Jul 11).

    Article  CAS  PubMed  Google Scholar 

  22. Cohen SJ, Stackman RW Jr. Assessing rodent hippocampal involvement in the novel object recognition task. A review. Behav Brain Res. 2015;285:105–17. https://doi.org/10.1016/j.bbr.2014.08.002 (Epub 2014 Aug 26).

    Article  PubMed  Google Scholar 

  23. Cohen SJ, Munchow AH, Rios LM, Zhang G, Asgeirsdóttir HN, Stackman RW Jr. The rodent hippocampus is essential for nonspatial object memory. Curr Biol. 2013;23(17):1685–90. https://doi.org/10.1016/j.cub.2013.07.002 (Epub 2013 Aug 15).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Izquierdo I, Barros DM, MelloeSouza T, de Souza MM, Izquierdo LA, Medina JH. Mechanisms for memory types differ. Nature. 1998;393(6686):635–6. https://doi.org/10.1038/31371.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

Supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil—Grant Number 407459/2021-9) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil—Code 001). The funding bodies did not play any role in the design of the study, the collection, analysis, and interpretation of data, or in writing the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

MCG and AR carried out the experiments, analyzed data, prepared the figures, and helped designing the experiments and drafting the manuscript. JIR participated in the behavioral studies. SCO developed some of the software routines utilized in the electrophysiological experiments. LRMB collaborated in the design and supervision of the study. MC conceived, planned, and supervised the study, analyzed data, designed the experiments, and wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Martín Cammarota.

Ethics declarations

Ethics approval and consent to participate

All experiments were performed following the NIH Guidelines for Animal Care and were approved by the Committee for the Ethical Use of Research Animals of the Federal University of Rio Grande do Norte (CEUA).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1.

Extended materials and methods and datasets.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gonzalez, M.C., Radiske, A., Rossato, J.I. et al. Optogenetic inactivation of the medial septum impairs long-term object recognition memory formation. Mol Brain 15, 50 (2022). https://doi.org/10.1186/s13041-022-00938-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13041-022-00938-3

Keywords

  • Theta rhythm
  • Amnesia
  • Hippocampus
  • Brain oscillations
  • Long-term memory