Functional neuroanatomy aims to understand the relationships between the wiring of neuronal networks and their function. The research of my group focuses on the architecture of the parahippocampal and hippocampal networks mediating learning and memory.

The central hypothesis for my research is that striking differences in functional properties of the lateral and medial entorhinal cortex emerge from subtle differences in intrinsic wiring combined with differences in input and output relationships.

1. Laminar and cellular terminal distribution of main inputs to the medial entorhinal cortex, such as inputs from the pre- and parasubiculum, the retrosplenial, parietal, and postrhinal cortices.
2. Laminar and cellular terminal distribution of inputs from orbital, medial prefrontal, and insular cortices to the lateral entorhinal cortex.
3. The comparative architecture of local networks of the lateral and medial entorhinal cortex.
4. The postnatal development of the cortico-parahippocampal-hippocampal network.

I accepted a professorship at the Kavli Institute in 2007, after having worked as a visiting member of CBM for 5 years. Since 2007, I have built up an active state-of-the-art neuroanatomy group of approximately 10 post-docs and PhD students. Over the years my work on the architecture of the entorhinal-hippocampal system carried out at VU University Medical Center Amsterdam, has led to a number of testable hypotheses that still are at the core of the ongoing quest to understand the functional contributions of the system to learning and memory processes.

In one of my first publications (Witter and Groenewegen, 1984), I postulated the concept of functional differentiation along the long axis of the hippocampus, which I further elaborated on in my 1989 review (Witter et al. 1989). This idea still strongly influences ongoing research. In a conceptual paper published in 2000, I summarized a body of work from my lab, substantiating the concept put forward in my 1989 paper of parallel processing of spatial and object information, mediated by the two subdivisions of the entorhinal cortex and the notion that these are differently processed in the different subdivisions of the hippocampus (Witter et al. 2000). This postulate eventually led to an influential series of collaborative papers with Edvard and May-Britt Mosers on grid cells.

We use advanced methodologies, including retrogradely and transsynaptically transported rabies virus that express different fluorophores, and multiple whole cell recordings (>3 cells) as a superior method to quantify local microcircuitry in the cortex in vitro. These approaches are complemented with a new advanced technique to express photo-inducible molecular channels such as channelrhodopsin-2 in specific input pathways, which allows to stimulate specific sets of axons that cannot be easily maintained or recognized in an in vitro slice preparation. This application builds on the group’s recently developed approach of in vivo tracing of input-output connectivity followed by in vitro recording. With the use of voltage sensitive dye imaging, we can assess efficiently whether the connectivity of interest is maintained in the slice, and we developed efficient methods to combine all these approaches. These new methods are complemented with traditional, yet powerful, anterograde and retrograde tracing of input and output pathways and newly developed confocal analyses in thick slices using sequential immunohistochemical staining procedures on intracellularly filled neurons as well as analyses at the electronmicroscopical level.

The functional anatomy toolbox. A. Two injections of rabies virus expressing different fluorophores in dorsal CA1. B. Single (red and green) and double labeled (yellow) retrogradely infected cells in layer III of LEC following injections shown in A (Ohara and Witter). C. In vitro patch of a neuron in layer III of MEC, receiving anterogradely labeled input from presubiculum. Left: low power image of section with injection site in PRS (yellow) and recorded and filled neuron (blue). Right: high power image of the same neuron which is clearly embedded in the labelled PrS axonal plexus; inset: response of layer III cell (Canto et al. 2012). D. Retrogradely labelled neurons in layers II and III of MEC following an injection in dorsal hippocampus (white/fluorogold) and in ventral hippocampus (blue/fast blue) in a P2 animal. E.Voltage sensitive dye imaging of presubicular activation of entorhinal cortex in adult rat (Koganezawa & Witter). F. Four layer II cells intracellularly filled with spectral variants of Alexa (red and green: stellate cells, blue and yellow: pyramidal cells (Couey et al. 2012). G. Sequential confocal analysis of synaptic connectivity of retrosplenial axons onto identified layer III neurons in presubiculum (Color code represents distance between elements of a potential synaps). Inset: bouton is characterized with the vesicular marker synaptophysin, postsynaptic element with PSD95 (Kononenko and Witter 2011). H. Single cell patch clamp recording of a layer V neuron in MEC showing EPSP’s evoked by laser stimulation of axons from the retrosplenial cortex infected with rAAVthat contained a mutant of the light-gated channelrhodopsin (ChiEF) together with mCherry as a fluorescent tag. Inset indicates stimulated area and evoked responses are on the left (Czajkowski et al, in prep).

In order to make our research data available to the scientific community, we have initiated a collaborative digital brain atlas on the parahippocampal-hippocampal region (Univ. Oslo; http://rbwb.org), and published a collaborative connectional database (http://www.temporal-lobe.com; van Strien et al., 2009, Nat Rev. Neurosci). The latter activities are embedded in the activities of the International Neuroinformatics Coordinating Facility (INCF; http://www.incf.org).

The research is embedded in active local interactions with May-Britt and Edvard Moser with respect to functional relevance of characteristic architecture, with Yasser Roudi regarding theoretical aspects of our observations and with incoming Cliff Kentros, currently at the University of Oregon, USA. We also have collaborations with a number of international investigators including Toshio Iijima, Tohoku Univ, Sendai, Japan, Yuchio Yanagawa, Gunma University Japan, Cliff Kentros, Claudio Cuello at McGill University, Montreal, Scott Small, Columbia Univ, and John Gigg, Univ Manchester, UK.

Translational promises are now also on the research agenda. While the primary goal of my research is to contribute to our understanding of cognition in the normal brain, the activity has considerable potential for translation to clinical applications. Because the entorhinal cortex is one of the first brain regions to be affected in patients with Alzheimer’s disease, functional insight into the underpinning entorhinal functions may ultimately provide clinical neurologists and health workers with essential tools for early diagnostics, prevention and treatment of Alzheimer’s disease. We have taken initiatives to explore this promising potential.

1. Van Strien NM. Cappeart N, Witter MP (2009) The anatomy of memory: An interactive overview of the parahippocampal-hippocampal network. Nature Rev Neurosci 10:272-282.
2. Ohara S, Inoue K, Witter MP, Iijima T. (2009) Untangling neural networks with dual retrograde transsynaptic viral infection. Front Neurosci, 3:344-349.
3. Langston RF, Ainge JA, Couey JJ, Canto CB, Bjerknes TL, Witter MP, Moser EI, Moser M-B. (2010) Development of the spatial representation system in the rat. Science 328:1576-1580.
4. Boccara, CN, Sargolini F, Thoresen VH, Solstad T, Witter MP, Moser EI, Moser M-B. (2010) Grid cells in pre-and parasubiculum. Nat Neurosci 13: 987-994
5. Kjonigsten LJ, Leergaard TB, Witter MP, Bjaalie JG (2011) Digital atlas of anatomical subdivisions and boundaries of the rat hippocampal region. Front. Neuroinform. 5:2. doi: 10.33 9/fninf.2011.00002.
6. Sugar J, Witter MP, van Strien NM, Cappaerts NLM (2011) The retrosplenial cortex: intrinsic connectiviuty and connections with the (para)hippocampal region in the rat. An interactive connectome. Front. Neuroinform. 5:7. doi: 10.3389/ fninf.2011.00007
7. Small, SA, Schobel SA, Buxton R, Witter MP, Barnes CA (2011) A pathophysiological framework of the hippocampal circuit in aging and disease. Nature Rev Neurosci, 12: 585-601.
8. Yartsev M, Witter MP, Ulanovsky N (2011) Grid cells without theta oscillations in the entorhinal cortex of bats. Nature, 479: 103-107.
9. Kononenko N, Witter MP (2012) Presubiculum layer III conveys retrosplenial input to the medial entorhinal cortex. Hippocampus 22: 881-895.
10. Canto CB, Witter MP (2012) Cellular properties of principal neurons in the rat entorhinal cortex. I. The lateral entorhinal cortex. Hippocampus, 22: 1256-1276.
11. Canto CB, Witter MP (2012) Cellular properties of principal neurons in the rat entorhinal cortex. II. The medial entorhinal cortex. Hippocampus, 22: 1277-1299.
12. Canto CB, Koganezawa N, Beed P, Moser EI, Witter MP. All Layers of Medial Entorhinal Cortex Receive Pre- and Parasubicular Inputs. J. Neurosci. In press