Functional Examination of a Reciprocal Neural Circuit between the Hypothalamus and Hippocampus – HYPO-HIPPO

The research proposed in this project aims to use a combination of recently developed methods to examine and better understand how the supramammillary nucleus (SuM), a hypothalamic region that is known to be active during reward and emotional-laden behaviors alters hippocampal activity. The SuM sends axonal projections to area CA2/CA3a and the dentate gyrus of the hippocampus. It is well established that the SuM plays a significant role in controlling the hippocampal theta rhythm, a phenomenon critical for memory formation. This project will use stereotaxic viral injection of viral vectors in combination with transgenic mouse lines in order to express light-activated channels in a precise location- and cell-specific way. Thus, light pulses can be used to selectively stimulate SuM fibers in the hippocampus, allowing for the direct examination of synaptic transmission for the first time. Using these strategies, the research in this proposal will have the following three goals. First, to elucidate the precise cellular targets of SuM fibers in the hippocampus, and to determine which neurotransmitters or neuropeptides are released. I will perform whole-cell recordings of principal cells and interneurons in the dentate gyrus and area CA2/CA3a in the hippocampus and activate SuM fibers with light. With this information, I will determine how SuM activity alters the output of hippocampal neurons at a cellular and network level. I will determine how the excitation of SuM fibers influences other intra- and extra- hippocampal inputs and the resulting effect this has on theta rhythm. Lastly, I aim to examine the reciprocal projection from area CA2 to SuM: to determine the post-synaptic targets, impact on activity and projection pattern of target cells. With similar methods, I will excite CA2 fibers in the SuM and examine the outcome in the hypothalamus.

1) The synaptic transmission of SuM input into area CA2/CA3a is both glutamatergic and peptidergic.
2) Long sequences of light pulses result in the release of substance P, which acts on the feed-forward transmission driven by schaffer collateral activity onto area CA2, and has no effect on distal CA2 inputs.
3) While there is a relatively meager glutamatergic transmission onto CA2 and CA3a pyramidal neurons, we have discovered a very robust excitatory connection onto interneurons in area CA2/CA3a.
4) This strong excitatory synaptic transmission of Sum input onto interneurons drives a large feed-forward inhibition onto pyramidal cells in areas CA2 and CA3.
5) Approximately half of the pyramidal cells that we recorded from and filled with biocytin (N=134 total) in area CA2 contain thorny excressences like CA3 pyramidal cells, indicating they receive input from granule cells of the dentate gyrus.
6) The interneurons that receive the strongest input from the SuM contain axons that project extensively to the pyramidal cell layer into area CA3. These cells have dentritic arbors and firing properties consistent with basket cells.

There is a wealth of information from in vivo experiments that have characterized the rhythmic network activity of the hippocampus and have found that oscillations such as the theta rhythm are very tightly linked to behavior. The generation of theta rhythm is attributed to two subcortical nuclei: the medial septum and the supramammillary nucleus (SuM). However, it is striking that even after several decades of research, the precise physiological mechanisms underlying this phenomenon are unknown.

By identifying the post-synaptic targets of SuM projections into the hippocampus, how SuM activity alters other hippocampal synapses and plasticity, and how the SuM is modulated by reciprocal connections with the hippocampus, this project will provide a significant advance in understanding the role of the this brain region in hippocampal activity. In addition, the SuM nucleus has been proposed to serve as a modulator of cognitive processes during strong emotional stimulation. Therefore, results obtained from the completion of this project will help to better understand the physiological processes that allow emotional context to alter memory formation.

Furthermore, while synaptic transmission through classical neurotransmitters has been extensively studied, the action of neuropeptides is much less understood. The release of neuropeptides has never before been performed with optogenetic methods. Previous work investigating neuropeptide release has relied on electrical stimulation and/or pharmacological approaches. Both of these methods lack the precise temporal and spatial control of photostimulation. The completion of this project will provide a foundation for examining neuropeptide release in the hippocampus as well as other regions in the nervous system. For instance, because Substance P is an important transmitter in pain perception these results will be relevant well beyond the hippocampal field.

1. sY Maury, Côme J, Piskorowski RA, Salah-Mohellibi N, Chevaleyre V, Peschanski M, Martinat C and Nedelec S. “Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes.” Nature Biotechnology. 2014 Nov 10. doi: 10.1038/nbt.3049.
2. K Nasrallah, Piskorowski RA, Chevaleyre V. “Inhibitory plasticity permits the recruitment of CA2 pyramidal neurons by CA3.” eNeuro. Jul 2015. DOI: 10.1523/ENEURO.0049-15.2015.
3. « Fixing the Memory Model » International Innovation, Issue 170, A Cerebral Matter. 12 January 2015.

The ability to form memories and recall past events is a profoundly important ability for many aspects and levels of life. In mammals, it is firmly established that the hippocampus is as essential structure for long-term memory formation. Furthermore, due to its relatively simple laminar organization, much has been learned about the physiological events that occur during memory formation. The current model of hippocampal information processing is a linear transfer of information from the cortex to three separate regions of the hippocampus, the dentate gyrus, area CA3 and area CA1. At each stage, incoming synaptic information is altered and processed as it is passed to the next segment. While this model can account for several learning processes, there are still major questions that demand a re-examination of the model. In addition to the cortex, brain regions that are very important in emotion and reward-related processes also send axons to the hippocampus. Exactly how these structures act to modulate the hippocampal circuit at the synaptic, cellular and network level is poorly understood. For decades is has been extremely difficult, if not impossible, to selectively activate one input without also activating other external and internal hippocampal projections. As a consequence, the physiological mechanisms underlying how emotional context alters memory formation are poorly understood. This is extremely relevant in the context of psychiatric disorders with emotional and cognitive impairments such as depression and schizophrenia.

The research proposed in this project aims to use a combination of recently developed methods to examine and better understand how the supramammillary nucleus (SuM), a hypothalamic region that is known to be active during reward and emotional-laden behaviors alters hippocampal activity. The SuM sends axonal projections to area CA2/CA3a and the dentate gyrus of the hippocampus. It is well established that the SuM plays a significant role in controlling the hippocampal theta rhythm, a phenomenon critical for memory formation. While the SuM-hippocampal projection has been investigated in vivo and at an anatomical level, several large and important questions remain. This project will use stereotaxic viral injection of viral vectors in combination with transgenic mouse lines in order to express light-activated channels in a precise location- and cell-specific way. Thus, light pulses can be used to selectively stimulate SuM fibers in the hippocampus, allowing for the direct examination of synaptic transmission for the first time.

Using these strategies, the research in this proposal will have the following three goals. First, to elucidate the precise cellular targets of SuM fibers in the hippocampus, and to determine which neurotransmitters or neuropeptides are released. I will perform whole-cell recordings of principal cells and interneurons in the dentate gyrus and area CA2/CA3a in the hippocampus and activate SuM fibers with light. With this information, I will determine how SuM activity alters the output of hippocampal neurons at a cellular and network level. I will determine how the excitation of SuM fibers influences other intra- and extra- hippocampal inputs and the resulting effect this has on theta rhythm. Lastly, I aim to examine the reciprocal projection from area CA2 to SuM: to determine the post-synaptic targets, impact on activity and projection pattern of target cells. It has recently been shown that CA2 neurons project to the SuM, indicating that the Sum-hippocampal network is reciprocal. With similar methods, I will excite CA2 fibers in the SuM and examine the outcome in the hypothalamus.