Despite the important diversity of interneurons, much attention is focused on parvalbumin-containing basket interneurons (PV+ interneurons), which is the largest population of interneurons in hippocampus. These are perisomatic-targeting interneurons and are strategically positioned to control pyramidal cell output since each of them can connect to as many as 2000-3000 principal cells. Recent studies report a significant change in theta and gamma rhythm, and a correlated deficit in learning and memory after altering PV+ interneurons in transgenic mice lines. Another popular type is the somatostatin-positive oriens lacunosum-moleculare interneurons (SOM+ O-LM). These are dendritic-targeting interneurons that can provide significant modulation of basal and apical dendrites of principal cells as well as gating of entorhinal input to principal cells.
Which type of interneurons has the capacity to generate theta rhythm? Modeling studies suggest that the relatively slow intrinsic firing properties and conductance of SOM+ O-LM interneurons would support theta generation while basket cells would contribute to faster frequency gamma oscillations. Thus, while it is widely agreed that interneurons are crucial for rhythm generation, it remains unknown how each subtype contributes to rhythm generation in either a primary or secondary role (i.e are passively entrained by pyramidal cells), and which interneurons can modulate the frequency or power of the theta rhythm.
In my lab, we want to determine the role of interneurons in theta generation in the hippocampal CA3, CA1 area and in the subiculum, the principal area for encoding and retrieval, by applying optogenetics and electrophysiology in our recently developed whole hippocampal and septohippocampal preparations in vitro. The role of interneurons in rhythm generation remains largely incomplete because, until recently, no tools were available to temporally control the activity of specific neuronal subtypes during network rhythms. The use of optogenetics to causally manipulate selective interneuron types will allow us for the first time to determine the role of interneurons in rhythm generation in the hippocampus.
The hippocampus is probably the most intensely investigated brain region. This is due to a number of factors such as its beautiful organization, a simplified yet quite elaborate connectivity and a role in a cognitive function, memory, which is of wide interest. A large proportion of the knowledge about hippocampal function has been obtained from hippocampal brain slices, a technique developed in the early 1970’s. Hippocampal slice experiments have been instrumental in revealing the intricate mechanisms underlying synaptic transmission and plasticity.
However, they have given limited insight into network activity generated through neural interactions because many of the connections between neurons are lost in such preparations leaving out many of the normal network activity. With these shortcomings in mind, our lab wanted to develop a preparation that had all the connections intact and where we could use state-of-the-art techniques commonly used in vitro. Inspired by a 1997 paper (Khalilov et al., 1997, Neuron) illustrating the technique of using whole hippocampus preparation in immature rats, our goal was to find the right experimental conditions to keep a complete hippocampus preparation of adult mice and rats in vitro.
Since 2008, we have used the complete mature hippocampus preparation and found that the hippocampus contains a variety of intrinsic self-generated theta and gamma oscillators. Therefore, this preparation offers a unique opportunity to better understand the basic principles for theta and gamma generation as well as the interactions between these oscillations. In addition to the whole hippocampus preparation, we also have developed the septohippocampal preparation which is a beautiful technique with both the medial septum and hippocampus are kept attached in vitro.
The isolated hippocampus in vitro with the CA3, CA1 and subicular regions in addition to the septal and temporal poles. We have developed a free-hand dissection technique to isolate the hippocampus or both the septum and hippocampus together.
Picture showing both the hippocampus (left) and the septum (right) with interconnections in vitro.
Diagram showing the theta recorded from the complete hippocampus in vitro and that recorded in vivo showing the surprising resemblance in the signal (adapted from Colgin and Moser’s News and Views in Nature Neuroscience, 2009)
Optogenetic modulation technology (“opto” for optical stimulation and “genetics” for genetically targeted cell types) is a method developed in 2006 by Karl Deisseroth at Stanford University that allows remote control of specific neural circuits with physiologically appropriate spatial and temporal resolution. Optogenetics, named in 2010 “scientific technique of the year” (Nature) and “breakthrough of the decade” (Science magazine), is a powerful tool to precisely manipulate in vitro and in vivo the activity of a given neuronal type embedded in a network using photostimulation.
When expressed in a genetically targeted neuronal population, the light-sensitive proteins Channelrhodopsin-2 (ChR2) variant ChETA from Chlamydomonas reinhardtii and the Archaerhodopsin (ArchT) from Halorubrum strain TP009, the two opsins we most often use, allow bimodal modulation of neuronal excitability (activation or inhibition, respectively) with millisecond timescale precision. ChETA is a monovalent cation channel that allows Na+ ions to enter the cell following exposure to ~473 nm blue light, whereas ArchT is a pump that removes positively charged protons from the inside (i.e hyperpolarizes the cell interior) that is activated upon illumination with ~580 nm yellow light. Their fast temporal kinetics (millisecond timescale) makes it possible to drive reliable trains of high frequency action potentials in vitro and in vivo using ChETA or suppress single action potentials within high frequency spike trains using ArchT.
A fiber optic-based system was developed in our lab that is appropriate for delivering light to both superficial and deep brain regions. Hence, optogenetics allows frequency-dependent interrogation of the medial septal neurons and hippocampal GABAergic interneurons in vitro and in vivo without inadvertent stimulation of neighboring neurons as occurs with electrical or chemical stimulation.
Picture showing set-up to perform electrophysiology and optogenetics. Here, blue light (to activate the opsin ChR2) is applied at different frequencies during theta rhythm to determine the role of activating specific types of interneurons
Optogenetic experiment showing responses to constant 500ms blue light (left) or 2ms pulses (right) in a recorded interneuron