The Effects of Inhibiting Neurons in Layer II of the Medial Entorhinal Cortex on Hippocampal Place Cells in CA1 and CA3

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1 The Effects of Inhibiting Neurons in Layer II of the Medial Entorhinal Cortex on Hippocampal Place Cells in CA1 and CA3 Roshan Chikarmane Kentros Lab A BIOCHEMISTRY MAJOR RESEARCH REPORT Presented to the Department of Chemistry & Biochemistry at the University of Oregon in fulfillment of the requirements for the degree of Bachelors of Science, May 2017 Clifford Kentros, Principal Investigator 0

2 ABSTRACT The hippocampus and medial entorhinal cortex (MEC) are brain regions important for spatial memory formation and retrieval. Problems with hippocampus and MEC underlay neurodegenerative disorders like Alzheimer s Disease and other forms of dementia. Therefore, it is important that the mechanisms by which these brain regions function and interact are elucidated. Neurons called place cells in the hippocampal CA1 and CA3 regions fire whenever an animal occupies a certain location, relative to landmarks in that local environment. The MEC Layer II (MEC-LII), a brain region that receives inputs from many regions of the cerebral cortex and projects directly into the hippocampus, also contains spatially responsive neurons called grid cells. While the behaviors of place cells and grid cells have been well characterized, it is unclear whether place cells utilize information from grid cells. This study will focus on how the activity of place cells in the CA1 and CA3 hippocampal regions respond to inhibition of MEC-LII neurons. To investigate this relationship, we will use a transgenic line of mice that expresses hm4 receptors exclusively in the MEC-LII. Administration of the hm4 ligand clozapine-n-oxide (CNO) decreases neuronal activity. Using this method, we can decrease MEC-LII activity and simultaneously measure the activity of place cells downstream in vivo while mice freely explore an environment. This experiment will measure the transfer of information between two important brain regions that give rise to learning and memory. 1

3 INTRODUCTION The hippocampus and medial entorhinal cortex (MEC) are brain regions important for the formation and retrieval of memory. Extensive clinical studies have shown that accumulation of neurofibrillary tangles, a hallmark of neuron loss in Alzheimer s Disease, occurs in the entorhinal cortex, CA1 hippocampal region, and CA3 hippocampal region during Stage I, Stage IV, and Stage V progressions, respectively. (Braak & Braak, 1991). Significant neuron loss in layer II of the MEC (MEC-LII) distinguishes even the mildest forms of Alzheimer s Disease from individuals without any forms of dementia (Gómez-Isla et. al. 1996). Therefore, a thorough understanding of how the hippocampus and MEC brain regions function and interact as a memory system is of paramount importance. Neurons in the hippocampal CA1 and CA3 regions called place cells fire whenever an animal occupies a specific location within a local environment, known as a place field (O Keefe & Dostrovsky, 1971). Populations of place cells have corresponding place fields that, in aggregate, represent the full area in a local environment, forming a systematic map of twodimensional space (O Keefe, 1976). A major portion of excitatory inputs into the hippocampus is comprised of neurons from the MEC-LII called grid cells, which are also spatially responsive. Each grid cell has multiple firing fields that are separated by a fixed distance and are arranged in a repeating triangular pattern that covers the local environment (Fyhn et. al., 2004). This gridlike arrangement has led some to suggest that grid cells are elements of a cognitive metric system for spatial navigation (Hafting et. al., 2005). Taken together, grid cells in the MEC-LII represent general two-dimensional space while hippocampal place cells represent specific locations that an animal occupies within that space. Curiously, spatial representations in CA1 and CA3 place cells persist after considerable lesioning of intrahippocampal networks, which has led many to believe that place cell activity may be generated by direct grid cell inputs from MEC-LII (McNaughton et. al., 1989; Brun et. al. 2002). Theoretical models illustrate how information from arrays of grid cells may combine to form place field representations in the hippocampus (O Keefe & Burgess, 2005; Fuhs & Touretzky, 2006; McNaughton et. al., 2006). However, there is currently a lack of empirical evidence that demonstrates the influence MEC-LII inputs have on CA1 or CA3 place cells. Recent findings in the Kentros Lab show that depolarization of MEC-LII neurons alter spatial representations in CA1 place cells (Kanter et. al. 2017). Place fields that occurred in one location prior to MEC-LII depolarization, shifted to a new location in CA1 place cells. Additionally, CA1 place cells showed increases in place field size and firing rate, with a conserved firing location, in response to depolarization of MEC-LII neurons. Interestingly, transformations in place field location have also been observed when salient extrinsic cues in a familiar environment have been moved and when the size or shape of the geometric borders have been altered (Muller & Kubie, 1987). This study aims to examine whether inhibition of MEC-LII neurons also causes changes in firing rate and firing location in CA1 and CA3 place cells. 2

4 RESULTS Transgenic Mouse Lines with MEC-LII Specific Control of Neuronal Activation In order to investigate how the activity of place cells in the CA1 and CA3 hippocampal regions respond to inhibition of MEC-LII neurons, it was necessary that a system be established to selectively inhibit neurons in Layer II of the MEC. This required experimental control over two factors: neuronal activity and expression specificity to the MEC-LII. To achieve experimental control over neuronal activity, we utilized a hm4di-teto line of transgenic mice that constitutively expressed hm4di Designer Receptors Exclusively Activated by Designer Drugs (DREAD) in the brain (Armbruster et. al. 2007). These modified muscarinic Gi-protein coupled receptors (GPCRs) are potently activated exclusively by the synthetic small molecule clozapine-n-oxide (CNO), which is pharmacologically inert in wild-type mice, to induce membrane hyperpolarization. This method is capable of efficaciously causing neuronal silencing upon intraperitoneal (IP) administration of CNO in live mice. To achieve expression profiles that are specific to the MEC-LII domain, we utilized a tta-ec driver line (Yasuda & Mayford, 2006). These mice strongly express a targeted gene primarily in layer II of the MEC as well as the pre- and para-subiculum regions, which project strongly into the superficial layers of the MEC but very weakly into the hippocampus (van Groen & Wyss, 1990; Honda & Ishizuka, 2004; Köhler, 1985). In contrast, expression ranges from very weak to not at all in neighboring regions including the deep layers of the MEC, the lateral entorhinal cortex, the visual cortices, and the caudal regions of the retrosplenial agranular cortex. We crossed the hm4di-teto DREADD line with the tta-ec driver line to yield doublepositive offspring that express hm4di DREADDs exclusively in the MEC-LII. Henceforth, we will refer to these offspring as hm4 mice. To visualize this localization, we performed in situ hybridization on coronal brain slices using an RNA probe that targeted hm4di mrna transcripts (Figure 1). In accordance with previous findings, hm4di expression is strong in MEC-LII neurons, but minimal in neighboring brain regions. Qualitatively, our expression profile matches that of Kanter et. al. (2017), who quantitatively determined that a similar expression scheme targeted approximately 27% of layer II stellate cells. Figure 1. Expression of hm4di DREADD receptors in transgenic mice limited to MEC-LII in right hemisphere of mouse brain in coronal slice. Visualized by in situ hybridization using hm4di-specific RNA probes that had previously been Nissl stained with cresyl violet. D, dorsal; L, lateral relative to midline; MEC-LII, layer II of medial entorhinal cortex; DREADD, Designer Receptors Exclusively Activated by Designer Drugs. Magnification is 3. Tissue viewed by Olympus BX61 microscope, BX-UCB control box, Prior ProScanIII motorized stage, and Lumen200Pro light source. Images captured by DP72 camera and processed in Photoshop CS4 (Adobe Systems, CA). 3

5 Screening and Experimental Protocols for Place Cell and Grid Cell Recordings To measure the activity of place cells we implanted multi-channel adjustable-depth tetrode arrays (four spun iridium/platinum filaments 18 microns in diameter with a polyamide coating and exposed ends) into the CA1 or CA3 regions of hm4 mice and allowed seven days of recovery prior to screening. A similar procedure was followed for the recording of grid cells, but the tetrodes were instead implanted into the MEC of hm4 mice. Each tetrode was secured in the channel of an EIB-16 electrode interface board, or drive, with gold pins, which made contact with a tethered HS-18MM operational amplifier during recordings of neuronal activity (Neuralynx, Bozeman, MT). Recordings in CA3 mice utilized a four-channel drive with a Teflon housing and three screws ( /8 ), which fixed the drive to the skull and facilitated the simultaneous adjustment of tetrode depth in all channels. Recordings in the CA1 and MEC-LII mice utilized a seven-channel VersaDrive-4 microdrive (Neuralynx) that facilitated the adjustment of tetrode depth in each individual channel. During screening sessions, implanted mice were allowed to freely roam a familiar environment. CA1 and CA3 mice roamed a circular environment with a diameter of 60 cm. Since the place field for place cells are known to orient relative to salient extrinsic cues that are arranged along a geometric boundary (Müller & Kubie, 1987), the cylindrical wall of the environment was black except for a white rectangle ( ) at the north end, which served as the dominant cue. MEC-LII mice roamed a cm square environment with black west, east, and south walls but a white north wall. A digital camera located above the environment recorded the position of the mouse by tracking the movements of two light-emitting diodes, oriented along the anteroposterior axis of the mouse. Concurrently, multi-unit neuronal activity was recorded and stored with the Cheetah- 16 system (Neuralynx). Potential changes within a certain range (minimum: 50 µv; maximum: µv) were automatically amplified and spikes were band-pass filtered ( Hz). Subsequent to recording, the activities of single units were manually separated with the MClust spike-sorting software for MATLAB using the selection criteria outlined in Kentros et. al. (2004). The same software was used to correlate place cell or grid cell activity with the mouse s position. Experimental sessions began with a 30-minute recording of baseline activity followed by an intraperitoneal injection of 10 mg/kg CNO (1.0 mg/ml in 10% DMSO/saline). Shortly after injection, the mouse was placed in the same environment and neuronal activity was recorded for 120 minutes. After hours, the mice were once again introduced into the same environment and data was collected for 30 minutes. 4

6 Mean Firing Rate (Hz) Inducible Hyperpolarization of Grid Cells in MEC-LII in hm4 Mice A cohort of hm4 mice with microelectrode implants in MEC-LII were first recorded for baseline activity (30 minutes) the cm square environment described in the previous subsection ( Screening and Experimental Protocols for Place Cell and Grid Cell Recordings ). The mice were then administered with 10 mg/kg CNO and were recorded in the same environment 120 minutes immediately following administration. Finally, hours after CNO, the mice were recorded once again for 30 minutes. Grid cells included in this analysis had a mean rate of less than 10 Hz and a peak firing rate of above 0.1 Hz. In the 120-minute recording session subsequent to CNO, grid cells in the MEC-LII showed a significant decrease in mean firing rate (hm4 MEC-LII n = 5, p < 0.001; ANOVA), although activity was restored to its baseline levels hours after CNO (Figure 2). These findings have two important implications for later experiments. Firstly, if 120-minute post-cno recordings are conducted on hm4 mice in the same time frame but, instead, place cells are recorded it can reliably be assumed that hyperpolarization is occurring in MEC-LII grid cells. Therefore, changes in place cell activity can be attributed to CNO-induced hyperpolarization of MEC-LII grid cells. Secondly, grid cell hyperpolarization is reversible, as seen by the restoration of mean firing rate to its basal level hours after CNO. Therefore, multiple place cell experiments can be conducted on the same mouse without disruption to baseline grid cell activity, as long as those experiments are separated by at least 12 hours. 4 * BL 0-30 min min min min +12 hr Figure 2. Administration of 10 mg/kg CNO reversibly induces decrease in mean firing rate in grid cells within MEC-LII. Mean ± SEM. *p <

7 Figure 3 shows the rate maps of representative grid cells in hm4 mice. These rate maps show a top-down view of the cm square environment with corresponding grid cell activity represented by different colors. Grid cells in the initial (Figure 3a) and latter (Figure 3b) portions of the 120-minute post-cno recording sessions show hyperpolarization, but show restoration of basal activity after 12 hours. Figure 3. Rate maps of representative MEC-LII grid cell shows that firing rate decreases in the (A) initial and (B) latter portions of the recording sessions following administration of CNO (10 mg/kg), relative to baseline. Time period following CNO injection and mean firing rate is represented above and below each rate map, respectively. Baseline rate maps occur prior to CNO injection. Red regions correspond to regions that the mouse occupied when the cell fired maximally, blue regions correspond to regions of neuronal silence, and white regions correspond to areas not explored by the mouse within the specified timeframe. 6

8 Normalized Rate Change Hyperpolarization of Neurons in MEC-LII Does Not Alter Firing Rate or Firing Location in CA1 or CA3 Place Cells A cohort of hm4 mice with microelectrode implants in CA1 or CA3 were first recorded for baseline activity (30 minutes) in the circular environment with a diameter of 60 cm described in the previous subsection ( Screening and Experimental Protocols for Place Cell and Grid Cell Recordings ). The mice were then administered with 10 mg/kg CNO and, immediately after administration, were recorded in the same environment for 120 minutes. Finally, hours after CNO, the mice were recorded once again for 30 minutes. Place cells included in this analysis had a mean rate between 0.1 and 7 Hz, a peak firing rate of above 1 Hz, and a spatial correlation value greater than 0.5 between the baseline and the hour session. Place fields were defined as having at least 20 contiguous pixels (80 cm 2 ) with a firing rate greater than 20% mean rate. As a precautionary measure, only the minute portion of the 120-minute post-cno time period was taken into account. Normalized rate changes were calculated as shown in Equation 1 below. Normalized Rate Change = Mean Rate Baseline Mean Rate min after CNO injection Mean Rate Baseline + Mean Rate min after CNO injection Neither CA1 nor CA3 place cells in hm4 mice showed changes in firing rate (Figure 4) or firing location (Figure 5) in response to hyperpolarization of MEC-LII cells, relative to wildtype mice (Wild-type CA1 n = 9, hm4 CA1 n = 33, hm4 CA3 n = 18; two-sample t-test). Their rate changes and spatial correlations did not vary significantly from the rate changes and spatial correlations seen in wild-type mice, which presumably did not experience depolarization in MEC-LII neurons following CNO. (1) Wild-type CA1 ( min) CA3 ( min) Figure 4. Hyperpolarization of MEC-LII neurons does not change mean firing rate in control group, CA1 and CA3 place cells. All groups hm4, except for Wild-type. Mean ± SEM. 7

9 Spatial Correlation Wild-type CA1 ( min) CA3 ( min) CA1 (+12 hr) CA3 (+12 hr) Figure 5. Hyperpolarization of MEC-LII neurons does not cause changes in firing location of place cells in control group, CA1 and CA3 place cells. All groups hm4, except for Wild-type. Mean ± SEM. Figure 6 shows the rate maps of representative place cells in hm4 mice. These rate maps show a top-down view of the circular environment (diameter 60 cm) with corresponding place cell activity represented by different colors. Place cells in the CA1 (Figure 6a) and CA3 (Figure 6b) hippocampal regions show mean rate and firing location similar to basal activity in the 120- minute session immediately after CNO. Figure 6. Rate maps of representative (A) CA1 place cell and (B) CA3 place cell show that firing rate and location remains constant following hyperpolarization of MEC-LII cells. Time period following CNO injection and mean firing rate is represented above and below each rate map, respectively. Baseline rate maps occur prior to CNO injection. Red regions correspond to regions that the mouse occupied when the cell fired maximally, blue regions correspond to neuronal silence, and white regions correspond to areas not explored by the mouse within the specified timeframe. 8

10 DISCUSSION This study demonstrates that, upon hyperpolarization of neurons in layer II of the medial entorhinal cortex, the firing rate and firing location of place cells in the CA1 or CA3 hippocampal regions remain relatively stable. Our outcome is, paradoxically, surprising because the MEC is a major source of excitatory inputs into the hippocampus (Zhang et. al., 2013), but expected because other studies report spatial tuning in CA1 place cells after extensive lesioning of the MEC (Hales et. al., 2014). Spatial tuning in the hippocampal CA1 and CA3 regions must be sustained through alternate parahippocampal inputs, which should be explored further. The stability of CA1 and CA3 place cells could also, conceivably, stem from the fact that hyperpolarization occurred in a fraction of MEC-LII neurons and that those neurons input to CA1 and CA3 place cells, which evaded detection. While MEC-LII inputs are not necessary for spatial tuning in hippocampal place cells, depolarization of MEC-LII inputs are sufficient to induce changes in CA1 place representations, namely changes in firing location and bidirectional changes in firing rate (Kanter et. al., 2017). However, we found that these changes do not occur when MEC-LII neurons are hyperpolarized. In combination, these findings serve a dual purpose. Firstly, they demonstrate that MEC-LII neurons and hippocampal place cells act as a functionally connected system rather than as independently functioning entities. Whether or not that system exclusively involves grid cells as the source of spatial modulation in place cells has yet to be determined, since other spatially selective cells reside in the MEC, namely border cells and head-direction cells (Sargolini et. al., 2006; Solstad et. al., 2006). Secondly, they clarify the mechanistic role of MEC-LII firing rate on spatial firing in the hippocampus. In a next step, we should record and include an analysis of border cell and head-direction cell hyperpolarization in response to CNO, as it would add nuance to the functional types of cell that are or are not necessary for spatial representations in CA1 or CA3. While it is not yet possible to isolate expression of inhibitory receptors, like hm4di, or excitatory receptors, like hm3dq, exclusively in grid cells, head-direction cells, or border cells without expression in surrounding tissues, it would be interesting to examine the effect of rate manipulation exclusively in those functional cell types on spatial representations in the hippocampus. 9

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