The reuniens nucleus of the thalamus facilitates hippocampo-cortical dialogue during sleep

Reviewer #1 (Public Review):

Summary:
In this study, Basha and colleagues aim to test whether the thalamic nucleus reuniens can facilitate the hippocampus/prefrontal cortex coupling during sleep. Considering the importance of sleep in memory consolidation, this study is important to understand the functional interaction between these three majorly involved regions. This work suggests that the thalamic nucleus reuniens has a functional role in synchronizing the hippocampus and prefrontal cortex.

Strengths:
The authors performed recordings in naturally sleeping cats, and analysed the correlation between the main slow wave sleep oscillatory hallmarks: slow waves, spindles, and hippocampal ripples, and with reuniens' neurons firing. They also associated intracellular recordings to assess the reuniens-prefrontal connectivity, and computational models of large networks in which they determined that the coupling of oscillations is modulated by the strength of hippocampal-thalamic connections.

Weaknesses:
The authors' main claim is made on slow waves and spindle coupling, which are recorded both in the prefrontal cortex and surprisingly in reuniens. Known to be generated in the cortex by cortico-thalamic mechanisms, the slow waves and spindles recorded in reuniens show no evidence of local generation in the reuniens, which is not anatomically equipped to generate such activities. Until shown differently, these oscillations recorded in reuniens are most likely volume-conducted from nearby cortices. Therefore, such a caveat is a major obstacle to analysing their correlation (in time or frequency domains) with oscillations in other regions.

Finally, the choice of the animal model (cats) is the best suited one, as too few data, particularly anatomical ones regarding reuniens connectivity, are available to support functional results.

Reviewer #2 (Public Review):

Summary:
The interplay between the medial prefrontal cortex and ventral hippocampal system is critical for many cognitive processes, including memory and its consolidation over time. A prominent idea in recent research is that this relationship is mediated at least in part by the midline nucleus reuniens with respect to consolidation in particular. Whereas the bulk of evidence has focused on neuroanatomy and the effects of temproary or permanent lesions of the nucleus reuniens, the current work examined the electrophysiology of these three structures and how they inter-relate, especially during sleep, which is anticipated to be critical for consolidation. They provide evidence from intercellular recordings of the bi-directional functional connectivity among these structures. There is an emphasis on the interactions between these regions during sleep, especially slow-wave sleep. They provide evidence, in cats, that cortical slow waves precede reuniens slow waves and hippocampal sharp-wave ripples, which may reflect prefrontal control of the timing of thalamic and hippocampal events, They also find evidence that hippocampal sharp wave ripples trigger thalamic firing and precede the onset of reuniens and medial prefrontal cortex spindles. The authors suggest that the effectiveness of bidirectional connections between the reuniens and the (ventral) CA1 is particularly strong during non-rapid eye movement sleep in the cat. This is a very interesting, complex study on a highly topical subject.

Strengths:
An excellent array of different electrophysiological techniques and analyses are conducted. The temporal relationships described are novel findings that suggest mechanisms behind the interactions between the key regions of interest. These may be of value for future experimental studies to test more directly their association with memory consolidation.

Weaknesses:
Given the complexity and number of findings provided, clearer explanation(s) and organisation that directed the specific value and importance of different findings would improve the paper. Most readers may then find it easier to follow the specific relevance of key approaches and findings and their emphasis. For example, the fact that bidirectional connections exist in the model system is not new per se. How and why the specific findings add to existing literature would have more impact if this information was addressed more directly in the written text and in the figure legends.

Author Response

eLife assessment

The manuscript presents valuable evidence of temporal correlations during specific oscillatory activity between the prefrontal cortex, thalamic nucleus reuniens, and the hippocampus, in naturally sleeping animals. Such correlations represent solid evidence to support the notion that the thalamic nucleus reuniens participates in the hippocampal and prefrontal cortex dialogue subserving memory processes.

Thank you for your assessment.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this study, Basha and colleagues aim to test whether the thalamic nucleus reuniens can facilitate the hippocampus/prefrontal cortex coupling during sleep. Considering the importance of sleep in memory consolidation, this study is important to understand the functional interaction between these three majorly involved regions. This work suggests that the thalamic nucleus reuniens has a functional role in synchronizing the hippocampus and prefrontal cortex.

Strengths:

The authors performed recordings in naturally sleeping cats, and analysed the correlation between the main slow wave sleep oscillatory hallmarks: slow waves, spindles, and hippocampal ripples, and with reuniens' neurons firing. They also associated intracellular recordings to assess the reuniens-prefrontal connectivity, and computational models of large networks in which they determined that the coupling of oscillations is modulated by the strength of hippocampal-thalamic connections.

Thank you for your positive evaluation.

Weaknesses:

The authors' main claim is made on slow waves and spindle coupling, which are recorded both in the prefrontal cortex and surprisingly in reuniens. Known to be generated in the cortex by cortico-thalamic mechanisms, the slow waves and spindles recorded in reuniens show no evidence of local generation in the reuniens, which is not anatomically equipped to generate such activities. Until shown differently, these oscillations recorded in reuniens are most likely volume-conducted from nearby cortices. Therefore, such a caveat is a major obstacle to analysing their correlation (in time or frequency domains) with oscillations in other regions.

1. We fully agree with the reviewer that reuniens likely does not generate neither slow waves nor spindles. We do not make such claim, which we clearly stated in the discussion (lines 319-324). We propose that Reuniens neurons mediate different forms of activity. In the model, we introduced MD nucleus only because without MD we were unable to generate spindles. While the slow waves and spindles are generated in other thalamocortical regions, the REU neurons show these rhythms due to long-range projections from these regions to REU as has been shown in the model.

2. Definitely, we cannot exclude some influence of volume conductance on obtained LFP recordings in REU nucleus. However, we show modulation of spiking activity within REU by spindles. Spike modulation cannot be explained by volume conductance but can be explained by either synaptic drive (likely the case here) or some intrinsic neuronal processes (like T-current).

3. In our REU recordings for spike identification we used tetrode recordings. If slow waves and spindles are volume conducted, then slow waves and spindles recorded with tetrodes should have identical shape. Following reviewer comment, we took these recordings and subtracted one channel from another. The difference in signal during slow waves is in the order 0.1 mV. Considering that the distance between electrodes is in the order of 20 um, such a difference in voltage is major and can only be explained by local extracellular currents, likely due to synaptic activities originating in afferent structures.

Finally, the choice of the animal model (cats) is the best suited one, as too few data, particularly anatomical ones regarding reuniens connectivity, are available to support functional results.

1. Thalamus of majority of mammals (definitely primates and carnivores, including cats) contain local circuit interneurons (about 30 % of all neurons). A vast majority of studies in rodents (except LGN nucleus) report either absence or extremally low (i.e. Jager P, Moore G, Calpin P, et al. Dual midbrain and forebrain origins of thalamic inhibitory interneurons. eLife. 2021; 10: e59272.) number of thalamic interneurons. Therefore, studies on other species than rodents are necessary, and bring new information, which is impossible to obtain in rodents.

2. Cats’ brain is much larger than the brain of mice or rats, therefore, the effects of volume conductance from cortex to REU are much smaller, if not negligible. The distance between REU and closest cortical structure (ectosylvian gyrus) in cats is about 15 mm.

3. Indeed, there is much less anatomical data on cats as opposed to rodents. This is why, we performed experiments shown in the figure 1. This figure contains functional anatomy data. Antidromic responses show that recorded structure projects to stimulated structure. Orthodromic responses show that stimulated structure projects to recorded structure.

Reviewer #2 (Public Review):

Summary:

The interplay between the medial prefrontal cortex and ventral hippocampal system is critical for many cognitive processes, including memory and its consolidation over time. A prominent idea in recent research is that this relationship is mediated at least in part by the midline nucleus reuniens with respect to consolidation in particular. Whereas the bulk of evidence has focused on neuroanatomy and the effects of temproary or permanent lesions of the nucleus reuniens, the current work examined the electrophysiology of these three structures and how they inter-relate, especially during sleep, which is anticipated to be critical for consolidation. They provide evidence from intercellular recordings of the bi-directional functional connectivity among these structures. There is an emphasis on the interactions between these regions during sleep, especially slow-wave sleep. They provide evidence, in cats, that cortical slow waves precede reuniens slow waves and hippocampal sharp-wave ripples, which may reflect prefrontal control of the timing of thalamic and hippocampal events, They also find evidence that hippocampal sharp wave ripples trigger thalamic firing and precede the onset of reuniens and medial prefrontal cortex spindles. The authors suggest that the effectiveness of bidirectional connections between the reuniens and the (ventral) CA1 is particularly strong during non-rapid eye movement sleep in the cat. This is a very interesting, complex study on a highly topical subject.

Strengths:

An excellent array of different electrophysiological techniques and analyses are conducted. The temporal relationships described are novel findings that suggest mechanisms behind the interactions between the key regions of interest. These may be of value for future experimental studies to test more directly their association with memory consolidation.

We thank this reviewer for very positive evaluation of our study.

Weaknesses:

Given the complexity and number of findings provided, clearer explanation(s) and organisation that directed the specific value and importance of different findings would improve the paper. Most readers may then find it easier to follow the specific relevance of key approaches and findings and their emphasis. For example, the fact that bidirectional connections exist in the model system is not new per se. How and why the specific findings add to existing literature would have more impact if this information was addressed more directly in the written text and in the figure legends.

Thank you for this comment. In the revised version, we will do our best to simplify presentation and more clearly explain our findings.