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Efficacy of nonselective optogenetic control of the medial septum over hippocampal oscillations: the influence of speed and implications for cognitive enhancement

2016; Wiley; Volume: 4; Issue: 23 Linguagem: Inglês

10.14814/phy2.13048

2051-817X

Benjamin J. Blumberg, Sean P. Flynn, Sylvain Barrière, Philippe R. Mouchati, Rod C. Scott, Gregory L. Holmes, Jeremy M. Barry,

Memory and Neural Mechanisms

Physiological ReportsVolume 4, Issue 23 e13048 Original ResearchOpen Access Efficacy of nonselective optogenetic control of the medial septum over hippocampal oscillations: the influence of speed and implications for cognitive enhancement Benjamin J. Blumberg, Benjamin J. Blumberg Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VermontSearch for more papers by this authorSean P. Flynn, Sean P. Flynn Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VermontSearch for more papers by this authorSylvain J. Barriere, Sylvain J. Barriere Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VermontSearch for more papers by this authorPhilippe R. Mouchati, Philippe R. Mouchati Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VermontSearch for more papers by this authorRod C. Scott, Rod C. Scott Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, Vermont Department of Neurology, Institute of Child Health, University College London, London, United KingdomSearch for more papers by this authorGregory L. Holmes, Gregory L. Holmes Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VermontSearch for more papers by this authorJeremy M. Barry, Corresponding Author Jeremy M. Barry jbarry4@uvm.edu Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, Vermont Correspondence Jeremy M. Barry, Department of Neurological Sciences, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405. Tel: (802) 656-4588 Fax: (802) 656-5678 E-mail: jbarry4@uvm.eduSearch for more papers by this author Benjamin J. Blumberg, Benjamin J. Blumberg Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VermontSearch for more papers by this authorSean P. Flynn, Sean P. Flynn Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VermontSearch for more papers by this authorSylvain J. Barriere, Sylvain J. Barriere Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VermontSearch for more papers by this authorPhilippe R. Mouchati, Philippe R. Mouchati Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VermontSearch for more papers by this authorRod C. Scott, Rod C. Scott Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, Vermont Department of Neurology, Institute of Child Health, University College London, London, United KingdomSearch for more papers by this authorGregory L. Holmes, Gregory L. Holmes Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, VermontSearch for more papers by this authorJeremy M. Barry, Corresponding Author Jeremy M. Barry jbarry4@uvm.edu Department of Neurological Sciences, University of Vermont College of Medicine, Burlington, Vermont Correspondence Jeremy M. Barry, Department of Neurological Sciences, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405. Tel: (802) 656-4588 Fax: (802) 656-5678 E-mail: jbarry4@uvm.eduSearch for more papers by this author First published: 06 December 2016 https://doi.org/10.14814/phy2.13048Citations: 12 Funding Information This study was funded by National Institute of Health grant NS073083 (GLH). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Optogenetics holds great promise for both the dissection of neural circuits and the evaluation of theories centered on the temporal organizing properties of oscillations that underpin cognition. To date, no studies have examined the efficacy of optogenetic stimulation for altering hippocampal oscillations in freely moving wild-type rats, or how these alterations would affect performance on behavioral tasks. Here, we used an AAV virus to express ChR2 in the medial septum (MS) of wild-type rats, and optically stimulated septal neurons at 6 Hz and 30 Hz. We measured the corresponding effects of these stimulations on the oscillations of the MS and hippocampal subfields CA1 and CA3 in three different contexts: (1) With minimal movement while the rats sat in a confined chamber; (2) Explored a novel open field; and (3) Learned and performed a T-maze behavioral task. While control yellow light stimulation did not affect oscillations, 6-Hz blue light septal stimulations altered hippocampal theta oscillations in a manner that depended on the animal's mobility and speed. While the 30 Hz blue light septal stimulations only altered theta frequency in CA1 while the rat had limited mobility, it robustly increased the amplitude of hippocampal signals at 30 Hz in both regions in all three recording contexts. We found that animals were more likely to make a correct choice during Day 1 of T-maze training during both MS stimulation protocols than during control stimulation, and that improved performance was independent of theta frequency alterations. Introduction The physiological capacity to temporally coordinate dynamic neural activity within and between neural networks is believed to underlie normal cognitive processes (Fenton 2015; Barry et al. 2016). Data supporting this theory largely stem from the demonstration that the temporal coordination of neuronal firing, with respect to theta oscillations within the hippocampal circuit (Mizuseki et al. 2009), is correlated with learning and memory (Robbe and Buzsaki 2009; Douchamps et al. 2013; Siegle and Wilson 2014; Barry et al. 2016). Specifically, both modeling and experimental work suggest that the dynamic phase relationships of synaptic current as well as the timing of action potentials during theta rhythm are critical in both encoding and retrieval by organizing the transfer of neural information between the hippocampus and neocortex and within the hippocampal circuit (Hasselmo 2005; Siegle and Wilson 2014). The temporal discoordination of CA1 place cell action potentials with respect to local theta oscillations was also found to correlate with learning deficits on a complex spatial task (Barry et al. 2016). Decades of research has begun to unravel the complex physiological mechanisms that underpin the generation of the theta rhythm along the septo-hippocampal axis (Green and Arduini 1954; Petsche et al. 1962; Freund and Antal 1988; Stewart and Fox 1989; Stewart and Fox 1990; Dragoi et al. 1999; Buzsaki 2002; Wang 2002; McNaughton et al. 2006aa; Vandecasteele et al. 2014; Bender et al. 2015, 2016; Mamad et al. 2015; Tsanov 2015; Gangadharan et al. 2016) that ultimately provides the temporal framework for the coordinated suppression or facilitation of synaptic inputs (Csicsvari et al. 1999) across the anatomical expanse of the hippocampal formation (Lubenov and Siapas 2009). Recent developments in the capacity to manipulate hippocampal oscillatory activity through optogenetic manipulation of the septal pacemaker (Laxpati et al. 2014; Vandecasteele et al. 2014; Bender et al. 2015; Mamad et al. 2015) demonstrate the potential of this approach to serve as both a powerful tool for addressing fundamental basic science questions regarding the role of theta in learning and memory (Buzsaki 2002; Hasselmo 2005) and function as a neuroprosthetic that generates artificial oscillations in those with learning and memory difficulties. However, before researchers can address these possible uses of novel technology, there are two critical obstacles that must be addressed. The first is that attempts to artificially generate oscillations have met with mixed results, depending largely on learning and memory demands (Turnbull et al. 1994; McNaughton et al. 2006aa; Shirvalkar et al. 2010; Lipponen et al. 2012) and few studies have examined how intrinsic oscillations might integrate with artificial oscillations in relation to current behavior (Bender et al. 2015). The second obstacle is that although cell-type selectivity in optogenetics has done much for the dissection of the septo-hippocampal circuit, this largely necessitates the use of transgenic mouse lines. As transgenic rat lines are still rare (Mamad et al. 2015), the method is largely unavailable to those that prefer rats as a model organism in cognitive research or for use in paradigms that require rat models (Iannaccone and Jacob 2009). Previous studies have shown that it is possible to entrain hippocampal oscillations through optogenetic control of the medial septum (MS) in wild-type rats (Laxpati et al. 2014), although the efficacy of this entrainment in relation to movement, which has been shown to be an important variable in cell-selective optogenetics (Vandecasteele et al. 2014; Bender et al. 2015), has not been shown. We therefore carried out several experiments to test the efficacy of nonselective optogenetic control of the medial septum in the entrainment of oscillations in both CA1 and CA3 subfields of the dorsal hippocampus while rats were in three different behavioral contexts: (1) Limited motor activity in a narrow ceramic chamber; (2) Freely exploring a novel open field; and (3) Learning a hippocampal-dependent memory task. In agreement with previous work, we show that nonselective optogenetic theta stimulation of the medial septum can clearly alter theta oscillations in the hippocampus. While the 1:1 entrainment of theta oscillations is possible during 6 Hz stimulation, at least in CA3, the stimulation interacts with ongoing intrinsic oscillations in a complex manner that appears to depend largely on the animal's mobility and speed. In contrast, gamma range optogenetic septal stimulation at 30 Hz was found to robustly increase the amplitude of corresponding frequency ranges in both hippocampal regions in all three recording contexts. Finally, we demonstrate for the first time that rather than interfering with memory processes, both stimulation protocols improve performance during training on a T-maze task independently of theta frequency alterations. Methods Overview Eight adult male wild-type Sprague–Dawley rats received medial septum injections of AAV (adeno-associated virus) that expressed channelrhodopsin. Two weeks post injection, rats were chronically implanted with an optic fiber with an attached recording electrode in the medial septum. Recording electrodes were implanted bilaterally in each hippocampus in both regions CA1 and CA3 (See Fig. 1). Following recovery, rats were stimulated with yellow light control and blue light frequencies at 6 Hz and 30 Hz while the animals either had limited mobility in a narrow ceramic chamber, explored a novel environment, or learned a T-maze behavioral task. Figure 1Open in figure viewerPowerPoint Methodology of AAV injection containing channelrhodopsin (ChR2) into the medial septum, optical stimulation, hippocampal, and medial septum electrophysiology: (A) ChR2 was delivered by AAV injection into the medial septum of adult rats. Expression of ChR2 in the pace-making cells of medial septum allows for the optical control of septal oscillations; (B) Arrangement of optical probe and EEG recording wires in the medial septum as well as EEG recording wires in both CA1 and CA3 fields of the dorsal hippocampus; (C) In response to blue light, ChR2-expressing neurons undergo a conformational change leading to the opening of cation channel pores and the conductance of positively charged ions such as Na+. The C-terminal end of ChR2 extends into the intracellular space and is replaced by yellow light-sensitive proteins (EYFP indicated by yellow blocks) that were used for visualizing the morphology of ChR2-expressing cells shown in D; (D) Histological example of forebrain tissue section under yellow light. Green fluorescence indicates cells in the medial septum expressing ChR2. All procedures were approved by the University of Vermont animal care and use committee and conducted in accordance with guidelines from the National Institutes of Health. Optical fiber preparation We used a 200-μm multimode optic fiber (Thorlabs, CFLC230-10; Montreal, Canada) as part of our chronic implant in order to allow for light stimulation of the medial septum. We stripped 25–35 mm of optic fiber using a microstripper. Using a razor blade, we stripped fiber from the main spool, leaving ~10 mm of unstripped fiber. We scored the end of the stripped fiber with a diamond knife on all sides. Hemostats were attached to opposite ends of the optic fiber and pulled apart. The piece of scored and stripped optic fiber then broke off cleanly. The optic fiber was then glued to a 230-μm ferrule (Thorlabs, CFLC230-10; TP01235931). First, the ferrule was placed in a vice with the convex side facing down. A droplet of dark epoxy (Precision Fiber Products, Epoxy 353ND/8 oz kit; Part A – PB117651), Part B PB117685) was then added to the larger (upward facing) end of the ferrule, so that there was an outward bubble. The remaining fiber covering was removed and the previously unstripped end was then placed through the epoxy, leaving 15 mm of fiber from the larger end of the ferrule. Using a heat gun, the dark epoxy was heated until cured (30 secs–1 min). The fiber was removed from the vice and the ferrule was held with a hemostat. All four sides of the fiber were then scored at the convex end of the ferrule using a diamond knife. To increase light transmittance, this cut end was polished using a fiber polishing kit (Thorlabs, Fiber polishing/lapping film for use with ceramic ferrules). Five different grades of polishing paper were used (30 μm, 6 μm, 3 μm, 1 μm, 0.02 μm grit) on a polishing glass plate/silicone pad (THORLABS, CTG913/NRS913A). While the ferrules were held with hemostats, the cut end of the fiber was polished at each grade of polishing paper. Finally, the percent transmittance of light through the fiber was tested using 100% blue light transmittance generated from Spectralynx light-emitting diode (LED) source and measured by a light meter with a photodiode sensor (Thorlabs; Model PM100D). Using a 50-μm patch cable for testing, only fibers that allowed for at least 70% light transmittance at approximately 0.5 mm from the tip of the optical fiber were used in our implants. Viral injection and chronic implantation surgeries Two separate surgeries were carried out on adult male Sprague–Dawley rats (n = 8) that were separated by 2 weeks. Rats were anesthetized with inhaled isoflurane and placed in a stereotaxic frame where all stereotaxic coordinates were relevant to Bregma (Paxinos and Watson 1998). Viral injection: The skull was exposed and a burr hole was placed in the skull (AP = 0.7 mm; ML = −1.4 mm) that allowed for targeting access of the vertical limb of the diagonal band of Broca of the medial septum with a Hamilton injection syringe (Fig. 1A). The syringe was inserted into the brain at 12° and placed at a final depth of 7.1 mm from brain surface. A total volume of 0.95 μL of adeno-associated virus expression of humanized ChR2 with H134R mutation fused to EYFP driven by human synapsin I promoter (AAV2-hSyn-hChR2(H134R)-EYFP; 5.7 × 1012 virus molecules/mL; UNC Vector Core, Chapel Hill, NC) was injected into the medial septum at a rate of 0.1 μL/min. The first injection of 0.15 μL was made at 7.1 mm. The needle was then retracted three times at 0.3 mm steps with injections of 0.2 μL, 0.25 μL, and 0.2 μL at each consecutive step. In the final step, the needle was raised 0.2 mm to a final depth of 6.0 mm and an ultimate injection of 0.15 μL was made. The wound was sutured and the rats were returned to their home cages to recover. Prior work has shown that AAV virus with the hSYN promoter nonselectively transfects different cell types in the medial septum (Chiruvella 2015). While a majority of the transfected cells were found to be glutamatergic or GABAergic (approximately, 70% of each cell type), a minority of cholinergic cells were also transfected (6%). Hippocampal and septal implants: Two weeks post injection, two custom electrode arrays (Grasshopper Machine Works, New Hampshire, USA) were implanted in both the medial septum and the dorsal hippocampus (Fig. 1B). The medial septum implant included an optic fiber with an array of eight recording electrodes glued to the surface that extended 0.25–0.5 mm from the end of the optic fiber. The optical/recording ensemble was lowered into the medial septum along the same path previously taken by the Hamilton injection syringe, with the end of the optical fiber lowered to a final depth of ~6.2 mm below brain surface. The hippocampal implant consisted of eight 50-μm diameter stainless steel EEG electrodes (California Fine Wire, CA, USA) that were placed in both the left and right hippocampus and were arranged so that medial electrodes targeted CA1 (AP = −3.7 mm; ML = ±2.5 mm; DV = 2.5–2.8 mm) and lateral electrodes targeted CA3 (AP = −3.7 mm; ML = ±3.8 mm; DV = 3.7–4.0 mm). Four skull screws (FHC Inc.) were inserted, two were anterior to bregma, while the two remaining screws were placed over the left and right of the cerebellum. Grounding was achieved via connection to the right cerebellar screw while a reference wire was placed through a small burr hole at brain surface over the cerebellum. Both implants were fixed to the skull via the skull screws and Grip Cement (Dentsply Inc.). The wound was sutured and topical antibiotic was applied. The interval between surgery and the beginning of electrophysiological recording was 1 week. Stimulation and recording protocols A 200-μm multimode optic fiber (Thor Laboratories, Budapest, Hungary) was used to connect the implanted optic fiber's 1.25 ceramic ferrule to Spectralynx, a computer-controlled optical LED system (Neuralynx, Montana, USA). Light intensity was set to 100% and transmitted intensity into the medial septum ranged from 1.4 to 1.8 mW. The pulse program (Neuralynx, Montana) was used to create protocols that generated square wave pulses at two stimulation frequencies of 6 Hz (on/off at 83.3 msec pulses) and 30 Hz (on/off at 16.6 msec pulses). We settled on 6 Hz and 30 Hz stimulation frequencies as pilot studies revealed that resultant matching hippocampal oscillations were visibly evident in the raw EEG. In addition, we chose these optical stimulation frequencies as they fall into functionally relevant hippocampal theta and gamma bandwidths. The theta frequency is the largest amplitude oscillation in hippocampus, associated with exploratory behavior and is principally generated by entorhinal cortical inputs into the distal apical dendrites of pyramidal cells (Buzsaki 2002). The waxing and waning of rhythmic inhibition corresponds to membrane potential fluctuations resulting in the temporal synchronization of neural activity on a ~140 msec timescale. The sequence of theta phase is therefore associated with the alternation of increased and decreased discharge probability of hippocampal neurons across the septotemporal axis (Fox 1989; Ylinen et al. 1995). In contrast, gamma oscillations are locally generated by the ongoing interaction of pyramidal cells and fast spiking interneurons in which pyramidal cells excite basket cells which then in turn inhibit pyramidal cells (Buzsaki and Wang 2012). Theta has been found to be necessary for spatial memory performance (Winson 1978) and is associated with the segmenting of spatial experience and modulated by behavior and cognitive demand (Gupta et al. 2012) as well as the formation and segregation of neuronal assemblies (Buzsaki 2002). Moreover, gamma oscillations have been hypothesized to "route" information flow in CA1 (Colgin et al. 2009) where slow gamma is more strongly coupled to CA3 and believed to be essential for memory storage (Steffenach et al. 2002). The rat's location in the arena was sampled using a digital camera that detected a LED placed near the animal's head. This tracking information was filtered and recorded utilizing custom software (Neuralynx, Montana, USA) that allowed for the synchronization of the rat's position and speed with properties of the recorded EEG signals. Rats were tethered to a recording cable during recording sessions in all contexts. LFP signals were preamplified ×1 at the headstage and channeled through the tether cable to the signal amplifiers and computer interface. LFPs (Local Field Potentials) were filtered at 1–9000 Hz (Neuralynx, Montana). All signals were referenced against a 50-μm diameter stainless steel wire (California Fine Wire, CA, USA) placed at brain surface over the cerebellum. Narrow ceramic chamber The purpose of the narrow chamber experiment was to test the efficacy of nonselective optogenetic septal control over hippocampal oscillations in the absence of exploratory movement or cognitive demand. Recording sessions were made while rats rested in a 40 cm high ceramic flower pot that was 27 cm wide at the base and lined with home cage bedding. The size of the ceramic pot limited the rat's movements to rearing and minor head movements and therefore limited the amount of theta oscillations associated with active exploration. Blue light stimulation protocols at 6 and 30 Hz consisted of five rounds of stimulations each. Each stimulation lasted 10 sec, with an interstimulus interval of 20 sec. Novel environment The purpose of the novel environment experiment was to test the efficacy of septal control over hippocampal oscillations during active exploration and examine the interaction of artificially generated oscillations with intrinsic oscillations. Animals were recorded for 20-min sessions during exploration of three consecutive novel environments. Stimulation protocols in the first environment consisted of yellow light control, while stimulation in the second and third novel environments consisted of blue light stimulation at 6 Hz and 30 Hz consecutively. Stimulations began as soon as the animals were introduced into the arena and continued until the end of the 20-min session. The same 76 cm diameter gray cylinder was used in three different rooms. A different cue card covering 60° of arc was used in each environment that consisted of a white card with black stripes in vertical, horizontal, or diagonal orientations. Similar protocols have been demonstrated to initiate complete remapping of hippocampal place cells, and by inference, considered by the rats to be novel environments (Leutgeb et al. 2005). T- maze The purpose of the T-maze alternation experiment was to test for the effects of MS optical stimulation on the acquisition and performance of a cognitive task. Following 1 week of food deprivation, the animals were trained on the T-maze alternation task (Deacon and Rawlins 2006). The T-maze consisted of a start box (20 cm × 15 cm), running lane (118 cm × 15 cm), and goal arms (137 cm × 15 cm). The animals were initially habituated in the T-maze without food reward and then placed in the maze with 20 mg food pellets (BioServ; Flemington, NJ) scattered throughout the maze. Food pellets were gradually moved to only the goal arms. Once the rats readily ran from the start box to the goal arms, three food pellets were placed in metallic food cups that were 3 cm in diameter and approximately 5 cm from the end of each goal arm. The animals were trained for 5–10 trials in order to be certain that they visited both arms. One goal arm was then randomly baited while access to the opposite arm was blocked. Once animals learned to go to both arms, they were considered ready for testing. During the sampling trial, the rat was first placed in the start box. The gate was opened and the rat ran to the end of the runway. For the first run, the rat was forced to go into the open arm with the food award. On the choice trial, both arms were open and the rat was rewarded with food if it went into the previously unrewarded arm. After reward, the animal was immediately returned to the start box. This sequence (sample and choice) was repeated for 30 test trials. Stimulations with 6 Hz yellow light and 6 Hz and 30 Hz blue were continuously applied from the start box to the completion of the task. The order of stimulations was randomized and consisted of 10 control, 10 6 Hz, and 10 30 Hz trials, so as no three stimulations of the same type were repeated in a row. The order of left and right choice directions was also randomly determined. The stimulation began before the start of the sample run and was not turned off until after the animal reached the end of a goal arm during the choice run. The animals were then retested in the T-maze. The first 30 trials on Day 1 of training were then compared to the 30 trials on Day 2 in order to test for the effects of stimulation on learning and memory. Signal processing Spectrograms were calculated with Matlab Spectrogram (window = SF/2, overlap = SF/2.4: SF = sampling frequency) and were used to calculate measures of signal amplitude and frequency at 5–12 Hz and 28–32 Hz bandwidths. In order to take into account the possibility of 6 Hz stimulation effects in a smaller theta bandwidth, we also analyzed "theta-lo" signal properties in the 5–7 Hz range. Mean spectrums For narrow ceramic chamber recordings, individual mean spectrums consisted of the mean of all spectrums for 10-sec epochs before stimulation and 10-sec epochs during stimulation. For novel environment recordings, data from the mean spectrum were calculated for the entire 10-min recording session during control yellow light and blue light stimulations at both 6 Hz and 30 Hz. Mean spectrums in blue light and yellow light control sessions were then compared using paired t-tests. With regard to recording sessions in the T-maze, mean spectral data were calculated for each pass the animal made through the center arm of the T-maze on both training days. As described below in the statistical methods, the spectral data for multiple runs were then clustered by individual rat. Peak-theta frequency Using a 1-Hz wide moving window (moving at 0.1 Hz), the average amplitude of theta in the 1-Hz window was calculated across the theta range (5–12 Hz) for each stimulation period. The midpoint of a 1-Hz moving window with the greatest average amplitude was designated as the peak-theta frequency and the average amplitude for that window was designated peak theta. Speed/theta We analyzed the linear relationship between animal speed in relation to theta frequency and amplitude in a similar manner to previous work (Richard et al. 2013) for data collected in the novel environment context. Statistical analyses As the behavioral and EEG dataset from narrow ceramic chamber and during the T-maze contain data from multiple behavioral choices or stimulations in single animals, the assumptions of independence of observations are invalid. The observations for each of these measures within single animals are likely to be correlated and these data can be represented as a cluster. In this case, the existence of a relationship between each measure of interest within an individual animal may then be assumed (Ziegler et al. 1998). In this study, we used GEE (SPSS; Armonk, NY), a class of regression marginal model, for exploring multivariable relationships between clustered signal property data for individual animals sorted by stimulation type in each recording context. At the behavioral level, we tested for differences in correct choices on both Day 1 and Day 2 during optical stimulation protocols of 6 Hz and 30 Hz. With regard to EEG, we tested the average response to the frequency and amplitude of oscillations in the theta range (5–12 Hz) as well as the amplitude of oscillations between 28 and 32 Hz during both 6-Hz and 30-Hz blue light optical stimulation protocols compared with yellow light controls or prestimulation epochs. Results Narrow ceramic chamber Seven rats were recorded in the narrow ceramic chamber during 6 Hz and 30 Hz blue light stimulation protocols of the medial septum. Responses in the medial septum and corresponding changes in the hippocampus were not found with two of these animals at either frequency. Histology showed that the optical fiber for one of these animals was misplaced while the optical fiber for the other rat had been damaged. These animals were therefore removed from analysis (N = 5). Key results from this experiment, including means and standard errors across subjects as well as the results of GEE analysis, are listed in Table 1. Table 1. Mean and standard error of EEG signal properties in the theta and slow gamma bandwidths in the narrow ceramic chamber Variable Condition CA1 CA1 P-value CA3 CA3 P-value Theta-lo freq. (5–7 Hz) Pre 6-Hz stimulation 6.20 ± 0.024 6.12 ± 0.030 During 6-Hz stimulation 6.04 ± 0.037 P = 0.001* 6.03 ± 0.014 P = 0.003* Theta freq. (5–12 Hz) Pre 6-Hz stimulation 7.10 ± 0.13 7.20 ± 0.13 During 6-Hz stimulation 7.35 ± 0.14 P = 0.105 6.78 ± 0.24 P = 0.006* Theta freq. (5–12 Hz) Pre 30-Hz Stimulation 7.14 ± 0.15 7.29 ± 0.16 During 30-Hz stimulation 7.62 ± 0.16 P = 0.012* 7.56 ± 0.17 P = 0.234 Theta amp. (5–12 Hz: A.U.) Pre 6-Hz stimulation 0.001016 ± 1.03 E-05 P = 0.362 0.000985 ± 1.36 E-05 P = 0.723 During 6-Hz stimulation 0.001005 ± 1.31 E-05 0.000983 ± 8.75 E-06 Theta amp. (5–12 Hz: A.U.) Pre 30-H