Effects of Selective M1 Muscarinic Receptor Activation on

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Effects of selective M muscarinic receptor activation on hippocampal spatial representations and neuronal oscillations Evan P. Lebois, John B. Trimper, Chun Hu, Allan I. Levey, and Joseph R. Manns ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00160 • Publication Date (Web): 01 Aug 2016 Downloaded from http://pubs.acs.org on August 7, 2016

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ACS Chemical Neuroscience

Effects of selective M1 muscarinic receptor activation on hippocampal spatial representations and neuronal oscillations Evan P. Lebois1,2, John B. Trimper3, Chun Hu4, Allan I. Levey5, and Joseph R. Manns3 1. Neuroscience Graduate Program, Emory University 2. Current Position: Neuroscience and Pain Research Unit, Pfizer Inc., Cambridge, MA 3. Department of Psychology, Emory University 4. Neuroscience and Behavioral Biology Program, Emory University 5. Department of Neurology, Emory University

Author Contributions: EPL, AIL, and JRM designed research; EPL, CH, and JBT conducted research; EPL, JBT, CH, and JRM analyzed data; EPL, JBT, CH, AIL, and JRM wrote manuscript. Conflict of Interests: EPL is a named inventor on the following patents and applications relevant to VU0364572: US Pat No 8,697,691; US Pat Appl 20130197027; US Pat Appl 20120088791. The other authors declare no conflicts of interest.

Address correspondence to: Joseph R. Manns, Ph.D. Associate Professor Department of Psychology Emory University 36 Eagle Row Atlanta, GA, 30322 [email protected] 404-727-7459

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Abstract The muscarinic M1 acetylcholine receptor is a key target for drugs aimed at treating cognitive dysfunction, including the memory impairment in Alzheimer’s disease. The overall question of the current study was to ask how systemic administration of the bitopic M1 agonist VU0364572, the M1 positive allosteric modulator BQCA, and the acetylcholinesterase inhibitor donepezil (current standard of care for Alzheimer’s disease), would impact spatial memory-related hippocampal function in rats. Hippocampal pyramidal neuron spiking and local field potentials were recorded from regions CA1 and CA3 as rats freely foraged in a recording enclosure. To assess the relative stability versus flexibility of the rats’ spatial representations, the walls of the recording enclosure were reshaped in 15-m intervals. As compared to the control condition, systemic administration of VU0364572 increased spatial correlations of CA1 and CA3 pyramidal neuron spiking across all enclosure shape comparisons, whereas BQCA and donepezil appeared to decrease these spatial correlations. Further, both VU0364572 and BQCA increased intra-hippocampal synchrony as measured by CA3-CA1 field-field coherence in frequency ranges that tended to align with the prominence of those oscillations for the behavioral state (i.e., theta during locomotion and slow gamma during stationary moments). The results indicated that VU0364572 and BQCA influenced hippocampal function differently but in ways that might both be beneficial for treating memory dysfunction. Key Words: muscarinic, acetylcholine, hippocampus, place cell, memory, oscillations


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Introduction Muscarinic acetylcholine receptors (mAChRs) are metabotropic G protein-coupled receptors that are found throughout the central and peripheral nervous systems 1-6. A large number of studies in rodents and humans have used systemic administration of muscarinic antagonists (e.g., scopolamine) to reveal the broad role of mAChRs in parasympathetic function, motor activity, and cognition 7-9. Further, in vitro studies using muscarinic agonists (e.g., carbachol) have highlighted a role for mAChRs in modulating synaptic plasticity and neuronal oscillations 7, 10-14. Five mAChRs subtypes have been identified, M1 through M5, and important differences exist across the subtypes in terms of where the receptors are found in the central and peripheral nervous systems and in terms of the details of signal transduction for each subtype 15, 16

. For example, activation of M1, M3, and M5 receptors leads to downstream increases in

intracellular calcium levels, whereas activation of M2 and M4 receptors leads to downstream inhibition of cyclic AMP. These differences in mAChR pharmacology are thought to have large implications for both understanding normal cholinergic function and for developing pharmacological treatments for a variety of disorders 15-17. In particular, drugs that can selectively activate one mAChR subtype have potential for improved efficacy and reduced adverse side effects as well as for avoiding the simultaneous engagement of opposing signal transduction cascades. The M1 receptor is a key target for new drugs aimed at treating cognitive dysfunction 1521

. Drugs that selectively activate the M1 receptor have potential for treating cognitive

dysfunction with fewer peripheral side effects as compared to non-specific muscarinic agonists because M1 receptors are almost exclusively expressed in the CNS relative to the periphery 1-6. In addition, in the brain, M1 receptors are heavily expressed in forebrain areas such as the



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neocortex and hippocampus that are linked to cognitive function 1-6. Further, transgenic mice lacking functional M1 receptors have shown cognitive deficits and, when combined with human Alzheimer’s disease-related mutations, markedly accelerated amyloid beta (Aβ) neuropathology 22-24

. Finally, selective M1 potentiators have been shown to exert anti-amyloidogenic effects on

amyloid-precursor protein (APP) processing both in vitro and in vivo in transgenic models of Alzheimer’s disease 23, 24. Thus, drugs that can selectively activate the M1 receptor are a promising route for new therapies to treat both the neurodegenerative and cognitive dysfunction (e.g., memory loss) observed in Alzheimer’s disease. A strategy of targeting allosteric and bitopic binding sites on the M1 receptor has led to the recent development of a number of selective M1 ligands 15-21. In particular, the bitopic M1 agonist VU0364572 has been shown to activate the M1 receptor with almost no activity across the other four mAChR subtypes 25. Another recently developed drug is BQCA, a positive allosteric modulator (PAM) that has also shown an extremely high degree of specificity for the M1 receptor 21. The pharmacokinetics of both drugs have been well-documented, including evidence for good brain penetration, and VU0364572 in particular has demonstrated excellent oral bioavailability. In rodent studies, VU0364572 has been shown to improve performance on the Morris water maze (MWM) and improve object recognition memory in rats 10, 26. Similarly, BQCA has been shown to improve performance on a reversal-learning task 27. Further, in tissue slices and cell cultures, VU0364572 and BQCA have been shown to increase pyramidal cell firing in hippocampus, mPFC, and striatum, induce hippocampal synaptic plasticity, to potentiate NMDAR currents, and to exert anti-amyloidogenic effects on APP processing 10, 23. Thus, much is known about the effects of these drugs at the cellular level and at the behavioral level. What is


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unknown is how systemic administration of selective M1 activators will impact neural activity in an awake and behaving animal. Decades of studies involving in vivo electrophysiological recordings from the rodent hippocampus have provided a rich characterization of the relationship between the rodent’s behavior and neural activity in this brain region that is not only key for memory but also influenced by widespread cortical and subcortical inputs 28. In particular, one of the best understood examples of brain-behavior relationships in neuroscience is the correlation between the rodent’s spatial location and the firing rate of individual hippocampal pyramidal neurons, neurons often called place cells due to their spatially-specific receptive fields 29. In addition, the hippocampus is one of the most studied regions for understanding the role of neuronal oscillations in synaptic transmission and synaptic plasticity 30. Furthermore, M1 receptors are expressed heavily in the hippocampus 4. Thus, the combination of selective M1 activators and hippocampal in vivo electrophysiology provides a great test bed for understanding how selective M1 activation will impact neural activity in the intact brain of an awake, behaving animal. The overall question of the current study was to ask how systemic administration of the bitopic M1 agonist VU0364572, the M1 positive allosteric modulator BQCA, and the acetylcholinesterase inhibitor donepezil (current standard treatment for Alzheimer’s disease), would impact spatial memory-related hippocampal function. Broadly speaking, three different types of cholinergic activation were examined: persistent-selective (M1 agonist VU0364572), phasic-selective (M1 potentiator BQCA), phasic-nonselective (pan-AChR activator donepezil). The agonist VU0364572 persistently activates M1 receptors independent of endogenous acetylcholine release, whereas BQCA activates M1 receptors only in the presence of phasicallyreleased endogenous acetylcholine.



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Hippocampal pyramidal neuron spiking and local field potentials were recorded from regions CA1 and CA3 as rats foraged for randomly scattered rewards in a recording enclosure. Figure 1 shows a schematic of the procedure as well as examples of spatial specificity in spiking data and oscillations in the local field potentials (see Methods for details). The objective was to use these well-characterized patterns of hippocampal activity to compare the influence of the drugs on a key brain region that would be both directly and indirectly influenced by activation of M1 receptors and that is key for normal memory. Several previous studies have highlighted the importance of acetylcholine and muscarinic receptors for normal hippocampal place fields and local field potential activity 31-34. However, it was unknown how activation of M1 receptors specifically would impact hippocampal activity in freely-locomoting rats. To obtain a more detailed understanding of how the drugs might influence spatial representations in the hippocampus, the walls of the recording enclosure were incrementally reshaped in 15-min recording periods within each session in order to alter the spatial input received by the hippocampus 35. Thus, one main question was how spatial correlates of hippocampal pyramidal neuron firing rates, one of the best-characterized neural correlates of spatial memory and behavior in rodents, would compare between enclosure shapes and how the drugs would impact both the within-shape and between-shape metrics of spiking activity. Another well-characterized neural correlate of rodent behavior is the relationship between locomotion and oscillations in the local field potentials as well as periodicity of spiking activity in the hippocampus. Thus, a second main question is how the drugs influenced oscillatory activity in the hippocampus during periods of movement and during stationary periods across all enclosure shapes in a session. Due to the relatively limited number of days during which consistently good recordings for each rat could be obtained, the present study focused on


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carefully chosen drug doses that had been shown in previous studies to yield behavioral efficacy. Results from basic measures of locomotion and neuronal activity (e.g., overall firing rates) are presented first, as the similarity of results in terms of these basic measures across drug conditions helps establish the specificity of drug effects for the subsequent results regarding spatial correlates and neuronal oscillations. The overall results indicate that selective M1 activation can influence hippocampal function in ways that could be beneficial for memory-impaired individuals. Results None of the drug conditions impacted basic measures of locomotion or hippocampal spiking Neural data were recorded across six testing sessions for each rat, the first and last of which were always sessions in which only control vehicles were administered (see Methods for details). The order in which the remaining four drug sessions (10 mg/kg VU0364572 p.o., 30 mg/kg VU0364572 p.o., 30 mg/kg BQCA s.c., and 3 mg/kg donepezil p.o.) were administered was randomized across rats. A total of 1,035 hippocampal pyramidal neurons were recorded (449 from region CA1 and 585 from region CA3). Table 1 shows the number of neurons recorded for each rat in each session. To minimize differences in tetrode positions across drug conditions, recording tetrodes were not moved between sessions. Although it was not possible to track individual neurons across days, the set of neurons recorded across days for each rat likely represented largely overlapping samples. Nevertheless, to minimize further the influence of any general changes in neural activity occurring over days (e.g., potential for the gradual drift in tetrode positions), all subsequent analyses compared the data from each drug session to the combined results from both control sessions.



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An initial question was whether any of the drug conditions influenced rats’ overt behavior, a result that, if observed, could have complicated subsequent analyses of neural data. Table 2 shows the mean percent of time in each session rats spent locomoting, the mean speed of locomotion when moving, and the mean percent of time spent near one of the enclosure walls (i.e., thigmotaxis). The rats’ overt behavior was similar across drug and control conditions. Separate one-way (across drug conditions, including the control condition) repeated-measures ANOVAs found no statistically significant effect of drug condition for percent time locomoting (F[4, 12] = 1.073; p = 0.412; partial η2 = 0.264), for locomotion speed (F[4, 12] = 0.584; p = 0.680; partial η2 = 0.163), or for percent time near walls (F[4, 12] = 0.336; p = 0.849; partial η2 = 0.101). The similarity of locomotive behavior across drug conditions indicates that any possible differences in neural activity across conditions were unlikely to be explained as indirect effects via an influence of the drugs on overt behavior. Mean firing rates of CA1 and CA3 pyramidal neurons were similar across drug and control conditions and are also shown in Table 2. A 2X5 (hippocampal region X condition) ANOVA (overall: F[9,1025] = 2.762; p = 0.003; partial η2 = 0.024) indicated that CA1 neurons had slightly yet statistically significantly higher firing rates in general than CA3 neurons (main effect of region: F[1, 1025] = 14.343; p = 0.00016; partial η2 = 0.02) but that firing rates did not significantly differ across drug conditions (main effect of condition: F[4, 1025] = 0.398; p = 0.810; partial η2 = 0.002). Moreover, direct contrasts between each drug condition and the control condition indicated that firing rates for none of the drug conditions differed significantly from control, even when the threshold for statistical significance was relaxed by not correcting the alpha level for multiple comparisons (all uncorrected ps > 0.3). Further, there was no statistically significant interaction between hippocampal region and drug condition (F[4, 1025] =


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1.576; p = 0.178; partial η2 = 0.006). Thus, none of the drug conditions appeared to impact appreciably the overall firing rates of CA1 or CA3 hippocampal pyramidal neurons. Moreover, the lack of an effect on firing rates differs from earlier studies in hippocampal slices or anesthetized animals using relatively high doses of scopolamine or carbachol but is consistent with recent studies that observed little influence on hippocampal firing rates or theta power of optogenetic stimulation of septal cholinergic neurons in locomoting rodents 36, 37. To ask if any of the drugs impacted in a general way the spatial correlates of pyramidal neuron spiking activity, a spatial information score was calculated for each neuron based on data from the square recording enclosure, combining data from both 15-min recording periods for each session. The spatial information score is a frequently used metric to assess the fidelity of the spatial representations in spiking activity 38. Mean spatial information scores of CA1 and CA3 pyramidal neurons were similar across drug and control conditions and are shown in Table 2. To ensure that there were enough spikes to calculate a meaningful spatial information score, only neurons with at least 500 spikes were used (total n = 361 and 374 for CA1 and CA3 regions, respectively). A 2X5 (hippocampal region X condition) ANOVA (overall: F[9,725] = 1.036; p = 0.409; partial η2 = 0.013) indicated that CA1 neurons had slightly higher spatial information scores in general than CA3 neurons (main effect of region: F[1, 725] = 5.376; p = 0.021; partial η2 = 0.007) but that spatial information scores did not significantly differ across drug conditions (main effect of condition: F[4, 725] = 0.556; p = 0.695; partial η2 = 0.003). Moreover, direct contrasts between each drug condition and the control condition indicated that spatial information scores for none of the drug conditions differed significantly from control, even when the threshold for statistical significance was relaxed by not correcting the alpha level for multiple comparisons (all uncorrected ps > 0.25). Further, there was no statistically



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significant interaction between hippocampal region and drug condition (F[4, 725] = 0.339; p = 0.852; partial η2 = 0.002). Thus, the above findings indicated that none of the drug conditions appeared to impact locomotion or the basic spiking metrics of hippocampal pyramidal neurons (overall firing rates and spatial information scores). These findings are important insofar as the results suggest that any subsequent differences between drug conditions are not due to differences in these basic measures. M1 agonist VU0364572 led to increased spatial correlations One of the main questions of the study was whether the drugs would influence how hippocampal spatial representations compared across shapes of the recording enclosures. To address this question, a spatial correlation was calculated for each pyramidal neuron between its firing in the initial square recording period and its firing in each of the subsequent recording periods when the walls of the enclosure were reshaped as a hexagon, octagon, circle, and, at the end, as a square again. Previous studies have shown this approach to provide a sensitive and informative indicator of hippocampal function and have suggested that the pattern of correlations might differ between CA1 and CA3 35, 39. Figure 2 shows an example from one CA3 pyramidal neuron recorded during a control session and highlights the general trend in the data that, as predicted, spatial correlations between the two periods in the square enclosure were higher than spatial correlations between the square enclosure and the other enclosure shapes. The question of interest was whether any of the drug conditions would alter the profile of spatial correlations between shapes relative to the control condition. For example, an increase in spatial correlations between the two periods in the square enclosure would suggest an increase in similarity of spatial representations, whereas an increase in spatial correlations between the square enclosure and circle enclosure would suggest reduced discriminability of spatial representations.


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Figure 3 shows the mean spatial correlations of CA1 and CA3 pyramidal neurons for the control condition and for each drug condition. To ensure that there were enough spikes to calculate a meaningful spatial correlation, only neurons with at least 500 spikes were used (total n = 361 and 374 for CA1 and CA3 regions, respectively). Some of those neurons did not emit spikes in each of the shape conditions, and thus the useable total number of units across conditions was reduced to 679 (n = 331 and 349 for CA1 and CA3 regions, respectively). For both CA1 and CA3 pyramidal neurons, the square-square mean spatial correlation was numerically higher in each condition than the mean spatial correlations between the square and other shapes of enclosures. However, as compared to the control condition, some of the drug conditions appeared to shift either up or down the overall level of spatial correlations. The general trend was that the 30 mg/kg VU0364572 condition resulted in overall higher spatial correlations, whereas BQCA and donepezil conditions resulted in overall lower spatial correlations. For example, for CA1 pyramidal neurons, the mean spatial correlations for all shape comparisons for the 30 mg/kg VU0364572 condition were the highest relative to the control and other drug conditions, whereas for the 3 mg/kg donepezil condition, the mean spatial correlations were the lowest for all shape comparisons. A 4X2X5 (shape comparison X hippocampal region X drug condition) general linear model was constructed in which the shape comparison was considered within units and the other two factors were considered between units (due to the inability to track individual neurons across multiple days). The results indicated that there was a statistically significant effect of shape comparison (F[3, 2007] = 69.806, p < .0001; partial η2 = 0.094), reflecting the trend of the square-square spatial correlation to be higher than the correlation between the square condition and each of the other shape conditions. The results also indicated that there was a statistically significant effect of drug condition (F[4, 669] = 3.996,



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p = .003; partial η2 = 0.023). The effect of hippocampal region (CA1 vs. CA3) was not statistically significant (F[1, 669] = 0.984, p = .322; partial η2 = 0.001). However, there was a statistically significant shape comparison by hippocampal region interaction (F[3, 2007] = 4.846, p = .002; partial η2 = 0.007). None of the other possible 2-way or 3-way interactions were statistically significant (all ps > 0.15). Thus, the main effect of the cholinergic drugs was to shift these spatial correlations overall higher or lower relative to the control condition. To ask more directly how the drugs influenced the spatial correlations relative to the control condition, the difference in spatial correlations between each drug condition and the control condition was calculated and is also shown in Figure 3 (bottom row). This approach sought to minimize variability between rats by comparing each rat’s data to its own control condition data in order to focus on effects of drug conditions (see Methods). Similar to the results for the absolute scores, these difference scores showed an effect of shape comparison (F[3, 1386] = 3.231, p = 0.022; partial η2 = 0.007) as well as an effect of drug condition (F[3, 462] = 5.952, p = 0.00055; partial η2 = 0.037). There was also a significant interaction between shape comparison and hippocampal region (F[3, 1386] = 5.511, p = 0.00092; partial η2 = 0.012). No other 2-way or 3-way interactions were statistically significant (all ps > 0.10). That is, the results suggest again that the effect of the drug conditions on spatial correlations relative to control conditions was an overall increase or decrease in spatial correlations. Specifically, combining across shape comparison and hippocampal region, the overall marginal mean difference (and 98.75% confidence intervals to correct for the four comparisons) from the control condition for the four drug conditions was 0.019 (-0.052 to 0.089), 0.102 (0.023 to 0.180), -0.054 (-0.126 to 0.018), and -0.49 (-0.118 to .019) for the 10 mg/kg VU0364572, 30 mg/kg VU0364572, 30 mg/kg BQCA, and 3 mg/kg donepezil conditions, respectively. These results


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suggest that whereas the BQCA and donepezil conditions resulted in overall lower spatial correlations, the most robust effect came from the overall increase in spatial correlations for the 30 mg/kg VU0364572 drug condition. In particular, the 30 mg/kg VU0364572 condition improved the extent to which two enclosures of the same shape were represented similarly, but it also increased the overlap in spatial representations of dissimilarly-shaped enclosures. The implication of this finding is considered further in the Discussion. M1 PAM BQCA altered pyramidal spiking relative to hippocampal theta rhythm The next question of interest was whether any of the drugs would influence the spiking of pyramidal neurons as it related to the ongoing theta oscillation in the hippocampus. The theta phase of spiking is known to be key to hippocampal function and can be influenced by cholinergic modulation of glutamatergic synaptic transmission in the hippocampus 12-14. As this question did not relate specifically to spatial representations but instead to active theta states, the neural data across all recording enclosure shapes were combined from periods when the rat was locomoting, which is when theta is known to be high in amplitude 25, 40-42. Prior to asking about spiking, we first asked if any drug condition influenced the frequency of the ongoing theta oscillations in the local field potentials. Table 3 shows for control and drug conditions the mean theta frequency of CA1 and CA3 pyramidal layer local field potentials during stationary periods and during periods of locomotion. Theta frequency was similar between each drug condition and the control condition (all drug vs. control contrasts resulted in p values greater than 0.1 even when no correction for multiple comparisons was used). Moreover, the relationship between theta frequency and running speed (see Table S1) and between theta power and running speed (see Table S2) was similar between each drug condition (see Supporting Information). Thus, any differences related to the theta phase of



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pyramidal neuron spiking are unlikely to be related to general changes in the frequency of the theta oscillations. Figure 4 shows for control and drug conditions measures of theta phase relationships between CA1 and CA3 pyramidal neuron spiking and hippocampal theta oscillations in the field potentials (as measured in the CA1 pyramidal layer to facilitate comparisons between regions and across rats). The peak, falling zero-crossing, trough, and rising zero-crossing of the theta oscillation in CA1 pyramidal cell layer was defined as 0, 90, 180, and 270 degrees, respectively. To ensure that there were enough spikes to calculate a meaningful mean phase for each neuron, only neurons with at least 500 spikes were used (total n = 361 and 374 for CA1 and CA3 regions, respectively). One specific question was whether the drug conditions would impact the mean theta phase at which CA1 or CA3 neurons tended to spike. The results for CA1 and CA3 neurons are plotted for each drug and control condition as circular means and circular 95% confidence intervals. The spiking of both CA1 and CA3 pyramidal neurons tended to be well-aligned with theta phase for the control and drug conditions (Rayleigh’s Z of mean phases across neurons for the control, 10 mg/kg VU0364572, 30 mg/kg VU0364572, 30 mg/kg BQCA, and 3 mg/kg donepezil conditions, respectively: CA1 = 47.97, 29.05, 18.84, and 27.86; CA3 = 7.66, 7.45, 6.52, 4.66, and 7.62; all ps 12.4 cm/s). As a result of this procedure, any difference in CA3-CA1 coherence between drug and control sessions would be unlikely to be explained by differences in speed of locomotion or in number of data segments. To evaluate the statistical significance of differences in CA3-CA1 coherence between each drug condition and the control condition, a cluster-based random permutation approach was used similar to that as described previously 56, 58. Specifically, for each drug-control contrast, the same resampling procedure described above was used again except that in each of the 10,000 samplings of the data, each 0.5 s segment of data was randomly assigned to the drug or control condition within each speed bin. The greatest drug-control difference was obtained from each random shuffle by identifying the cluster (i.e., above-threshold frequency range) with the largest absolute sum of coherence differences that exceeded +1 or -1 standard deviations across frequencies. Above-threshold clusters in the original data were then compared to the distribution of these randomly-generated maximum clusters, and a drug-control coherence difference was labelled as being statistically significant if the original cluster size was in the top or bottom 2.5 percentile of the random distribution. The advantage of the cluster-based procedure is that it



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does not calculate statistical significance for each individual frequency and thus does not inflate the overall alpha rate by performing multiple comparisons. Theta phase was obtained using a waveform-based method similar to that used previously 56

. The theta phase at which each spike occurred when the rat was locomoting was then obtained

for each CA1 and CA3 pyramidal neuron. The peak, falling zero-crossing, trough, and rising zero-crossing of the theta oscillation in CA1 pyramidal cell layer was defined as 0, 90, 180, and 270 degrees, respectively. For pyramidal neurons emitting at least 500 spikes across the shape conditions, several metrics of spike-phase relationships were calculated using an open-source circular statistics toolbox for MATLAB 59. These metrics included the circular mean of spike phases for each neuron as well as the circular phase mean and 95% circular confidence interval across neurons in a session. In addition, the mean resultant vector length, one measure of the degree of periodicity in spiking, was calculated for each neuron.


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Table 1. Number of pyramidal neurons (CA1 + CA3) recorded per session Rat

Control 1

1

57 (24+33)

VU0364572 (10 mg/kg) 68 (22+46)

VU0364572 (30 mg/kg) 40 (12+28)

BQCA

Donepezil

Control 2

Total

52 (18+34)

77 (25+52)

47 (12+35)

341 (113+228)

2

20 (2+18)

14 (3+11)

15 (1+14)

18 (1+17)

11 (0+11)

13 (6+7)

91 (13+78)

3

60 (49+11)

54 (32+22)

57 (35+22)

59 (30+29)

43 (31+12)

28 (11+17)

301 (188+113)

4

37 (13+24)

53 (31+22)

44 (17+27)

51 (16+35)

66 (39+27)

51 (19+32)

302 (135+167)

Total

174 (88+86)

189 (88+101)

156 (65+91)

180 (65+115)

197 (95+102)

139 (48+91)

1035 (449+586)

Table 2. Neuronal and locomotive measures: means (standard deviations) Control VU0364572 VU0364572 BQCA (10 mg/kg) (30 mg/kg) 0.55 (0.79) 0.57 (0.87) 0.64 (1.17) 0.55 (0.83) Firing Rate 0.75 (0.86) 0.66 (0.97) 0.73 (1.17) 0.81 (1.05) CA1 0.39 (0.70) 0.48 (0.78) 0.58 (1.17) 0.41 (0.64) CA3

Donepezil 0.54 (0.93) 0.55 (0.71) 0.53 (1.10)

Spatial Info CA1 CA3

1.90 (1.69) 1.68 (1.38) 2.14 (1.95)

1.98 (2.07) 1.96 (2.23) 2.00 (1.90)

1.94 (1.86) 1.79 (1.82) 2.07 (1.91)

1.71 (1.96) 1.42 (1.08) 1.93 (2.38)

1.95 (2.12) 1.75 (2.26) 2.17 (1.96)

% Moving Run speed % Thigmotaxic

27.7 (12.3) 18.9 (2.8) 60.8 (12.1)

21.0 (7.6) 18.1 (2.7) 63.0 (13.6)

25.7 (11.0) 18.1 (2.8) 64.3 (17.0)

21.5 (8.1) 18.4 (2.9) 60.1 (14.9)

25.1 (10.6) 18.5 (2.7) 61.4 (12.5)



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Table 3. Theta Frequency in Hertz: means (standard deviations) Control VU0364572 VU0364572 BQCA (10 mg/kg) (30 mg/kg) Stationary 8.26 (0.13) 8.26 (0.12) 8.27 (0.09) 8.31 (0.17) CA1 8.45 (0.08) 8.41 (0.12) 8.43 (0.06) 8.45 (0.06) CA3

8.26 (0.10) 8.43 (0.08)

Moving CA1 CA3

8.75 (0.25) 8.65 (0.16)

8.75 (0.16) 8.67 (0.14)

8.78 (0.20) 8.70 (0.02)

8.74 (0.20) 8.67 (0.11)

8.75 (0.17) 8.68 (0.11)


Donepezil

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Acknowledgments We thank the Vanderbilt Center for Neuroscience Drug Discovery for providing VU0364572 and BQCA. We also thank Matthew Hamm for his assistance conducting the research. This work was supported by funds from Emory University and by NIH Training Grant T32NS007480-12 (EPL).



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Contents of Supporting Information: 1. Table S1. Slope of Theta Frequency by Locomotion Speed 2. Table S2. Slope of Theta Power by Locomotion Speed 3. Figure S1. Power of CA1 local field potentials for drug and control sessions. 4. Figure S2. Power of CA3 local field potentials for drug and control sessions. 5. Figure S3. Recording tetrode positions for each rat.


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Figure Captions Figure 1. Experimental procedure. A. Overview of each testing session. Rats were administered a drug or control vehicle 30-40 min prior to a session in which neural data were recorded from the hippocampus as they foraged for randomly scattered chocolate-flavored pellets in an enclosure in which the walls could be reshaped (see Method for details). Rats spent 15 min in each shape condition in an order indicated by ordinal numbers. The enclosure shapes are arranged left-to-right according to the geometric similarity of the shapes. B. Example histology. Action potentials from individual neurons and local field potentials were recorded in both CA1 and CA3 subregions of the hippocampus. Circles indicate marking lesions made at the tips of recording tetrodes in both regions. C. Example spiking data from a CA3 pyramidal neurons during a 5-s period foraging during a control session. Rats’ paths (shown here as a dashed black line) were tracked during foraging and firing rates of action potentials (shown here as red dots) from individual hippocampal pyramidal neurons were analyzed as a function of the rat’s spatial position within the enclosure. D. Example local field potential data recorded simultaneously from CA1 and CA3 for the same 5-s period shown in panel C. Large theta oscillations at approximately 8 Hz and smaller gamma oscillations at approximately 40 Hz are visible in the recordings. The data were analyzed to quantify levels of oscillatory synchrony (e.g., coherence) between CA1 and CA3 as well as spike-field relationships (e.g., theta phase of spiking) during periods of foraging and during periods when the rat was stationary. The same action potentials from panel C are replotted here as red dots. Scaling of time and voltage are indicated by scale bars.



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Figure 2. Example color-coded spatial firing rate maps and spatial correlations for the same example hippocampal neuron shown in Figure 1. Data are from a control session. For each 15min period in which the recording enclosure was reshaped, the spiking activity of individual hippocampal pyramidal neurons was represented as an average firing rate (spikes per second) for each pixel (spatial bin). Many neurons, often termed place cells, showed a high degree of spatial specificity within a shape in their firing rates. The consistency of the pattern of firing rates between enclosure shapes was assessed for each neuron by calculating a (z-transformed) pixelby-pixel spatial correlation. The example spatial correlations shown illustrate the general trend in which the spatial correlation between the two periods in the square-shaped enclosure was higher than the spatial correlation between the first square-shaped enclosure and any of the other shapes.

Figure 3. Spatial correlations between recording enclosure shapes of CA1 and CA3 pyramidal neurons for drug and control sessions. A. The mean across neurons of z-transformed spatial correlations are plotted for each drug session and for the mean of two control sessions in which only control vehicles were administered. The correlations are plotted across comparisons between the first and second square-shaped enclosures (sq/sq) and between the first square and each of the other shapes (sq/hex, sq/oct, sq/circ). Higher correlations indicate greater similarity in the spatial pattern of firing rates. B. The differences between each drug condition and the control condition are plotted across the same shape comparisons shown in panel A. Error bars show SEM across neurons. The legend shows line colors for the mean control condition and for each drug condition (10 mg/kg VU0364572 p.o., 30 mg/kg VU0364572 p.o., 30 mg/kg BQCA s.c., and 3 mg/kg donepezil p.o.).


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Figure 4. Theta phase of CA1 and CA3 pyramidal neuron spiking for drug and control sessions. A. Mean phase of spiking of CA1 and CA3 pyramidal neurons relative to hippocampal theta oscillations (as recorded in the CA1 pyramidal layer) for drug and control sessions. For each circular plot, the colored wedge indicates circular mean (direction of solid line in the center of the wedge) and circular 95% confidence interval (width of the colored wedge). The peak (P), falling (F), trough (T), and rising (R) phases of the theta oscillation are indicated on the plots for the control sessions. The assignment of colors to drug conditions (10 mg/kg VU0364572 p.o., 30 mg/kg VU0364572 p.o., 30 mg/kg BQCA s.c., and 3 mg/kg donepezil p.o.) is provided above the circular plots and is maintained in subsequent panels. B. Mean theta phase difference for CA1 and CA3 pyramidal neurons between spiking in drug and control sessions (drug-control). Asterisks indicate statistically significant differences between a drug condition and the control condition. Error bars show SEM. C. Mean Resultant Length (MRL) of spiking theta phases for CA1 and CA3 pyramidal neurons. Error bars show SEM. Higher values indicate a greater alignment of spiking to the theta oscillations in the local field potentials. D. Difference in MRL between each drug condition and the control mean. Asterisks indicate statistically significant differences between a drug condition and the control condition. Error bars show SEM.

Figure 5. Coherence between CA3 and CA1 local field potentials for drug and control sessions. A. Mean CA3-CA1 coherence for control sessions during periods when the rat was stationary and moving. Peaks in coherence are visible in the theta range and in the low gamma range for both stationary and moving periods. The dark center line indicates the mean and the shaded area surrounding the mean indicates SEM. B. Mean difference in CA3-CA1 coherence between each



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of the drug conditions and the control condition (drug – control). The dark center line on each plot indicates the mean and the shaded area surrounding the mean indicates SEM. The horizontal bars with asterisks indicate frequency ranges that differed statistically significantly from the control condition. The labels indicate line colors for the mean control condition and for each drug condition (10 mg/kg VU0364572 p.o., 30 mg/kg VU0364572 p.o., 30 mg/kg BQCA s.c., and 3 mg/kg donepezil p.o.).


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Graphical Abstract

Hippocampal Place Cell

M1 Muscarinic Bitopic Agonist VU0364572

Control Vehicle

ACS Paragon Plus Environment

M1 Muscarinic Positive Allosteric Modulator BQCA


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A Drug or Control Vehicle

Square

Hexagon

Octagon

Circle

Square

1st

3rd

2nd

4th

5th

15 min

15 min

15 min

15 min

15 min

B

CA1

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C

CA3

D CA1

CA3 1s


1 mV

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Square

Hexagon

Octagon

Circle

Square 15 Hz 0

spatial correlation = 1.34 spatial correlation = 1.50



A

Spatial Correlations Between Shapes

Spatial Correlation

0.7

B

0.7

CA1

0.7

CA3

0.6

0.6

0.6

0.5

0.5

0.5

0.4

0.4

0.4

0.3

0.3

0.3

0.2

0.2

0.2

0.1

Spatial Correlation

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sq/sq sq/hex sq/oct sq/circ

0.1

0.1


Combined


Drug - Control Difference 0.3

0.3

CA1

0.3

CA3

0.2

0.2

0.2

0.1

0.1

0.1

0

0

0

-0.1

-0.1

-0.1

-0.2


-0.2

Comparison Control

-0.2


Comparison

VU0364572(10)

VU0364572(30)


Combined


Comparison BQCA

Donepezil

A

Control

VU0364572 VU0364572 10 mg/kg

CA1

F

T

30 mg/kg

BQCA

Donepezil

P

CA3

R F

P

T

B 75

.25

CA1

.25

.20

.20

.15

.15

.10

.10

.05

.05

0

0

CA3

D .05 MRL Difference

C

CA1

75

50

50

25

25

0

0

-25

-25

*

-50

R

Mean Resultant Length (MRL)

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Phase Difference (degrees)

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CA1

.05

0

-.05

-.05


*

*

-50

0

-.10

CA3

-.10

CA3


A Coherence

Stationary

1.0 0.5 0.0

Moving

1.0 0.5

20

40

60

80

100

0.0

20

40

60

80

100

Drug - Control Difference

Coherence Coherence Coherence

B

Stationary

0.15 0.10

VU0364572(10)

*

0.05 0.00

0.10 0.00

0.15

0.15

0.10

VU0364572(30)

0.10

0.05

0.05

0.00

0.00

-0.05

-0.05

0.15

0.15

BQCA

*

0.05 0.00

VU0364572(10)

0.05 -0.05

0.10

Moving

0.15

-0.05

**

VU0364572(30)

*

*

0.10 0.05

BQCA

0.00 -0.05 0.15

-0.05

Coherence

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Control CA3-CA1 Coherence

Page 48 of 48

0.15

Donepezil

0.10

*

0.05 0.00

Donepezil

0.10 0.05 0.00 -0.05

-0.05 20

40

60

80

Frequency (Hz)

100

20

40

60

80

Frequency (Hz)


100

Effects of Selective M1 Muscarinic Receptor Activation on

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