Analyzing Clustered Data: Why and How to Account for Multiple Observations Nested within a Study Participant?

A conventional study design among medical and biological experimentalists involves col- lecting multiple measurements from a study subject. For example, experiments utilizing mouse models in neuroscience often involve collecting multiple neuron measurements per mouse to increase the number of observations without requiring a large number of mice. This leads to a form of statistical dependence referred to as clustering. Inappropriate analy- ses of clustered data have resulted in several recent critiques of neuroscience research that suggest the bar for statistical analyses within the field is set too low. We compare naïve ana- lytical approaches to marginal, fixed-effect, and mixed-effect models and provide guidelines for when each of these models is most appropriate based on study design. We demonstrate the influence of clustering on a between-mouse treatment effect, a within-mouse treatment effect, and an interaction effect between the two. Our analyses demonstrate that these sta- tistical approaches can give substantially different results, primarily when the analyses include a between-mouse treatment effect. In a novel analysis from a neuroscience per- spective, we also refine the mixed-effect approach through the inclusion of an aggregate mouse-level counterpart to a within-mouse (neuron level) treatment as an additional predic- tor by adapting an advanced modeling technique that has been used in social science research and show that this yields more informative results. Based on these findings, we emphasize the importance of appropriate analyses of clustered data, and we aim for this work to serve as a resource for when one is deciding which approach will work best for a given study

Fatty Acids Increase Neuronal Hypertrophy of Pten Knockdown Neurons

Phosphatase and tensin homolog (Pten) catalyzes the reverse reaction of PI3K by dephosphorylating PIP3 to PIP2. This negatively regulates downstream Akt/mTOR/S6 signaling resulting in decreased cellular growth and proliferation. Co-injection of a lentivirus knocking Pten down with a control lentivirus allows us to compare the effects of Pten knockdown between individual neurons within the same animal. We find that knockdown of Pten results in neuronal hypertrophy by 21 days post-injection. This neuronal hypertrophy is correlated with increased p-S6 and p-mTOR in individual neurons. We used this system to test whether an environmental factor that has been implicated in cellular hypertrophy could influence the severity of the Pten knockdown-induced hypertrophy. Implantation of mini-osmotic pumps delivering fatty acids results in increased neuronal hypertrophy and p-S6/p-mTOR staining. These hypertrophic effects were reversed in response to rapamycin treatment. However, we did not observe a similar increase in hypertrophy in response to dietary manipulations of fatty acids. Thus, we conclude that by driving growth signaling with fatty acids and knocking down a critical regulator of growth, Pten, we are able to observe an additive morphological phenotype of increased soma size mediated by the mTOR pathway

Cognitive Deficits Associated with Na(v)1.1 Alterations: Involvement of Neuronal Firing Dynamics and Oscillations

Brain oscillations play a critical role in information processing and may, therefore, be essential to uncovering the mechanisms of cognitive impairment in neurological disease. In Dravet syndrome (DS), a mutation in SCN1A, coding for the voltage-gated sodium channel Na(v)1.1, is associated with severe cognitive impairment and seizures. While seizure frequency and severity do not correlate with the extent of impairment, the slowing of brain rhythms may be involved. Here we investigate the role of Na(v)1.1 on brain rhythms and cognition using RNA interference. We demonstrate that knockdown of Na(v)1.1 impairs fast-and burst-firing properties of neurons in the medial septum in vivo. The proportion of neurons that fired phase-locked to hippocampal theta oscillations was reduced, and medial septal regulation of theta rhythm was disrupted. During a working memory task, this deficit was characterized by a decrease in theta frequency and was negatively correlated with performance. These findings suggest a fundamental role for Na(v)1.1 in facilitating fast-firing properties in neurons, highlight the importance of precise temporal control of theta frequency for working memory, and imply that Na(v)1.1 deficits may disrupt information processing in DS via a dysregulation of brain rhythms

DREADDs: Use and Application in Behavioral Neuroscience

Technological advances over the last decade are changing the face of behavioral neuroscience research. Here we review recent work on the use of one such transformative tool in behavioral neuroscience research, chemogenetics (or Designer Receptors Exclusively Activated by Designer Drugs, DREADDS). As transformative technologies such as DREADDs are introduced, applied, and refined, their utility in addressing complex questions about behavior and cognition becomes clear and exciting. In the behavioral neuroscience field, remarkable new findings now regularly appear as a result of the ability to monitor and intervene in neural processes with high anatomical precision as animals behave in complex task environments. As these new tools are applied to behavioral questions, individualized procedures for their use find their way into diverse labs. Thus, "tips of the trade" become important for wide dissemination not only for laboratories that are using the tools but also for those who are interested in incorporating them into their own work. Our aim is to provide an up-to-date perspective on how the DREADD technique is being used for research on learning and memory, decision making, and goal-directed behavior, as well as to provide suggestions and considerations for current and future users based on our collective experience. (PsycINFO Database Recor

DREADDS: Use and application in behavioral neuroscience.

Technological advances over the last decade are changing the face of behavioral neuroscience research. Here we review recent work on the use of one such transformative tool, chemogenetics (or Designer Receptors Exclusively Activated by Designer Drugs, DREADDS), in behavioral neuroscience research. As transformative technologies such as DREADDs are introduced, applied, and refined, their utility in addressing complex questions on behavior and cognition become clear and exciting. In the behavioral neuroscience field, remarkable new findings now regularly appear as a result of the ability to monitor and intervene in neural processes with high anatomical precision as animals behave in complex task environments. As these new tools are applied to behavioral questions, individualized procedures for their use find their way into diverse labs. Thus, “tips of the trade” become important for wide dissemination not only for laboratories that are using the tools but also those that are interested in incorporating them into their own work. Our aim is to provide an up-to-date perspective on how the DREADD technique is being used for research on learning and memory, decision making, and goal-directed behavior, as well as to provide suggestions and considerations for current and future users based on our collective experience

Subcellular localization impacts PTEN activity in the murine dentate gyrus

One out of 36 children is diagnosed annually with Autism Spectrum Disorder in the United States. Loss of function mutations in PTEN characterize PTEN Hamartoma Tumor Syndrome, a multi-system group of syndromes associated with increased risk of Autism Spectrum Disorder and increased risk of breast, thyroid, and renal cancer. One possibility to support development of targeted therapies for those with PTEN mutations is to better understand the mechanisms within commonly mutated pathways. In this study, we examine subcellular localization of PTEN and how it may affect the neuronal morphology of cells. Mechanistically, PTEN dephosphorylates PIP3 to PIP2,thereby lowering downstream AKT activation and downregulating the mTOR complexes in the P13K / AKT / mTOR pathway in the cytosol, post-synaptic density, and cytoskeleton. Within the nucleus, PTEN functions as transcriptional regulator of p53 and various checkpoints in the cell cycle, operating independently of its phosphatase activity. The P13K / AKT / mTOR pathway is largely implicated in the growth and division of cells and is highly conserved in healthy cells to regulate neuronal soma size and other growth characteristics. As demonstrated by our lab’s previous research, loss of PTEN as a negative regulator of the P13K / AKT / mTOR pathway results in increased soma size and dendritic branching in hippocampal granule neurons. This work will expand on previous research which demonstrates in mice that PTEN knockout results in neuronal overgrowth by investigating how the subcellular localization of PTEN affects its regulation of neuronal morphology via retroviral-mediated recombination of four PTEN fusion proteins (NES-PTEN, NLS-PTEN, PTEN-FBAR, and PTEN-Homer) in order to achieve spatial control over PTEN. PTEN subcellular localization was measured using PTEN fluorescence intensity ratios in various subcellular compartments relative to other subcellular compartments and the background. In this thesis, we demonstrate success at optimizing PTEN immunostaining methodology in somas for all constructs except for PTEN-FBAR and show that PTEN-Homer and NLS-PTEN have respectively localized PTEN to the cytosol and the nucleus. Only PTEN-Homer out of all four fusion proteins achieved detectable PTEN immunostaining greater than background in dendrites and spines. Because control PTEN over-expression could not be detected above background in dendrites we were unable to determine if subcellular localization of PTEN in dendrites and spines was altered by our fusion proteins. In addition, we uncovered unexpected PTEN immunostaining in NES-PTEN mimicking that of PTEN knockout while maintaining a wildtype (WT) morphology. This poster will ponder these conclusions and brainstorm the next steps in examining other techniques for subcellular localization of PTEN.https://digitalcommons.dartmouth.edu/wetterhahn_2023/1002/thumbnail.jp

Reduced phase-locking of MSDB units to hippocampal theta rhythm.

<p>(A,B) Example of hippocampal LFP and theta-filtered signal with the simultaneously-recorded spike train from one MSDB unit. Notice the alignment (phase-locking) of spikes near the peak of the hippocampal theta oscillation. (C) Example of phase histogram of spikes in a standard (left) and rose plot (right) showing a strong theta phase preference. In left plot, red line indicates phase of hippocampal theta, and black bars indicate the phase of spikes. In right plot, phase histogram is shown in blue, theta phase as coordinates on the rose plot, and the red line indicates the mean resultant vector. (D) Histograms of theta phase-locking strength (mean resultant vector length) for units from controls and shScn1a rats. Phase-locking strength and (E) the proportion of units with statistically-significant phase-locking (Rayleigh test) were reduced after knockdown of Na_v1.1 (p<.01).</p

Impaired spatial working memory on a T-maze after MSDB Na_v1.1 knockdown.

<p>(A) Schematic of T-maze apparatus used for working memory task. Rats were trained to run from the start box to the end arm to receive a food reward. On each test trial, rats were given a Sample run (a forced turn either left or right), followed by a variable delay period, and then a Choice run. Correct choices required the rat to remember and recall the direction it had travelled on the Sample run and to then choose the opposite direction on the Choice run. (B) Mean choice accuracy for all trials was significantly worse with knockdown of Na_v1.1 compared to controls (p<.01). (C) Choice accuracy segregated into 24-trial blocks. Performance was initially lower for both groups during ‘variable delay’ trials, but performance improved in controls and not in shScn1a rats. (D) Choice accuracy segregated by delay length (short, 15-30s; long, 60s). While performance was lower overall for shScn1a rats, they were substantially worse at the longest delay, performing no better than chance levels. Data show mean +/- SEM. *p<.05, **p<.01.</p

Suppression of hippocampal theta rhythm after MSDB Na_v1.1 knockdown.

<p>(A) Schematic of recording site. (B) Example of LFP signal from the dorsal hippocampus of a control (black) and shScn1a (red) rat. (C) Group averaged power spectra for control and shScn1a rats. Inset shows power in the theta band. Solid line indicates mean, dashed line indicates SEM. (D-F) Theta frequency and theta power were significantly reduced in shScn1a rats, but no change in high frequencies in the gamma band were observed. (G) shScn1a rats also spent less time in theta. (H) Group averaged time-frequency spectrograms for 30s of LFP data time-locked to tail-pinch events. Notice that a prominent theta rhythm is apparent in response to the tail pinch but occurs at a lower frequency in shScn1a rats. (I,J) Quantification of theta frequency and theta power for data in panel H. Bars represent mean +/- SEM. *p<.05, **p<.01.</p