The Laboratory of Cellular Neurobiology
Currently our lab consists of Jennifer Luebke (PI), Graduate Students Josh Gilman, Jingyi Wang & Teresa Guillamon-Vivancos, & Senior Postdoctoral Fellow, Maya Medalla.
Confocal laser scanning microscope image of a typical mouse frontal cortical layer 3 pyramidal cell which was filled with biocytin during whole-cell patch-clamp recording and subsequently labeled with Alexa-Streptavidin-488
Dr Luebke‘s laboratory is interested in the electrophysiological and morphological properties of neocortical neurons in the rodent and primate prefrontal cortex across the lifespan. Our current work on neocortical pyramidal cells is divided into 5 research areas:
- Normative intrinsic membrane properties (e.g. action potentials and ionic currents).
- Normative glutamatergic and GABAergic synaptic response properties.
- Normative detailed morphological properties (e.g. dendritic architecture and spines).
- The effects of normal aging on the above properties in the rhesus monkey.
- The effects of beta-amyloid and tau on the above properties in transgenic mouse models of Alzheimer’s disease and other tauopathies.
Pyramidal Cells of the Prefrontal Cortex.
Working memory, which is essential for abilities such as abstract thinking, problem solving, and cognitive flexibility, is mediated in large part by pyramidal cells of the prefrontal cortex (PFC). We are interested in determining the basic properties of layer 2/3 and layer 5 pyramidal cells in both young and aged monkeys and in transgenic mouse models of Alzheimer’s disease. We employ whole-cell patch clamp and intracellular dye-filling techniques to examine the detailed properties of individual pyramidal cells in in vitro slices (see Images below).
We perform experiments on brain tissue obtained both from behaviorally characterized rhesus monkeys (as part of an integrated program project) and from transgenic mouse models of Alzheimer’s disease (such as the APP mutant Tg2576 and the tau mutant Tg4510). While we have experience in recording from many brain areas (hippocampus, brainstem areas, diverse neocortical areas), our research is currently focused on the prefrontal cortex- a brain area essential for higher cognitive function. The prefrontal cortex is removed from the experimental subject’s brain as a block and then cut into 400 micron thick living slices which are maintained in oxygenated artificial cerebrospinal fluid for up to 15 hours.
Whole-Cell Patch Clamp Recordings in In Vitro Brain Slices.
Using infrared differential interference contrast microscopy, we visualize living neurons in the in vitro slices and use whole-cell patch-clamp methodologies to record the electrophysiological and pharmacological response properties of identified neurons. We employ current-clamp techniques to examine action potential firing properties and intrinsic properties (such as resting membrane potential, input resistance and membrane time constant), and voltage-clamp techniques to examine ionic currents, responses to pharmacologic agents (primarily neurotransmitter agonists) and synaptic response properties. In addition, we provide single cells (from which recordings are obtained) to the Abraham laboratory for single cell PCR and microarray analyses, and filled neurons to the Peters laboratory for electronmicroscopy.
Morphometric Analyses of Neurons.
At the same time that recordings are obtained, we fill the neurons with intracellular dyes such as biocytin or Lucifer yellow. In addition, we fill neurons in fixed slices obtained from the same subjects. Dye-filled neurons are then scanned at ultra-high resolution using confocal laser scanning microscopy. Very detailed analyses of dendritic, somatic and axonal architecture are then undertaken. Dendritic spine morphology, number and density are also assessed. All morphological data are then correlated with electrophysiological data from the same neurons.
The intersection of amyloid beta and tau in glutamatergic synaptic dysfunction and collapse in Alzheimer’s disease. Crimins JL, Pooler A, Polydoro M, Luebke JI, Spires-Jones TL. Ageing Res Rev. 2013 Mar 22. doi:pii: S1568-1637(13)00015-9. 10.1016/j.arr.2013.03.002. [Epub ahead of print]intersection_of_amyloid_beta
The antiaging protein Klotho enhances oligodendrocyte maturation and myelination of the CNS. Chen CD, Sloane JA, Li H, Aytan N, Giannaris EL, Zeldich E, Hinman JD, Dedeoglu A, Rosene DL, Bansal R, Luebke JI, Kuro-o M, Abraham CR. J Neurosci. 2013 Jan 30;33(5):1927-39. doi: 10.1523/JNEUROSCI.2080-12.2013.
Dendritic spine changes associated with normal aging. Dickstein DL, Weaver CM, Luebke JI, Hof PR. Neuroscience. 2012 Oct 13. doi:pii: S0306-4522(12)01009-3. 10.1016/j.neuroscience.2012.09.077. [Epub ahead of print]dendritic_spine
Influence of highly distinctive structural properties on the excitability of pyramidal neurons in monkey visual and prefrontal cortices. Amatrudo JM, Weaver CM, Crimins JL, Hof PR, Rosene DL, Luebke JI. J Neurosci. 2012 Oct 3;32(40):13644-60. doi: 10.1523/JNEUROSCI.2581-12.2012.influence
Electrophysiological changes precede morphological changes to frontal cortical pyramidal neurons in the rTg4510 mouse model of progressive tauopathy. Crimins JL, Rocher AB, Luebke JI. Acta Neuropathol. 2012 Dec;124(6):777-95. doi: 10.1007/s00401-012-1038-9. Epub 2012 Sep 14.electrophysiological
Morphologic evidence for spatially clustered spines in apical dendrites of monkey neocortical pyramidal cells. Yadav A, Gao YZ, Rodriguez A, Dickstein DL, Wearne SL, Luebke JI, Hof PR, Weaver CM. J Comp Neurol. 2012 Sep 1;520(13):2888-902. doi: 10.1002/cne.23070.morphologic_evidence
Crimins JL, Rocher AB, Peters A, Shultz P, Lewis J, Luebke JI (2011) Homeostatic responses by surviving cortical pyramidal cells in neurodegenerative tauopathy. Acta Neuropath. 2011 Oct 4. [Epub ahead of print]
Kopeikina, K, Carlson G, Pitstick R, Ludvigson A, Peters A, Luebke J, Koffie R, Frosch M, Hyman B, Spires-Jones T, (2011) Tau accumulation causes mitochondrial distribution deficits in neurons in a mouse model of tauopathy and in human AD brain. Am J Pathol. 179(4): 2071-2082.
Ludvigson, A, Luebke, JI, Lewis, J, Peters A (2010) Structural abnormalities in the cortex of the rTg4510 mouse model of tauopathy: a light and electron microscopy study. Brain Structure and Function. 216(1):31-42.
Luebke, J.I., Weaver, C.M., Rocher, A.B., Rodriguez, A., Crimins, J.L., Dickstein, D.L., Wearne, S.L., Hof, P.R. (2010) Dendritic vulnerability in neurodegenerative disease: insights from analyses of cortical pyramidal neurons in transgenic mouse models. Brain Structure and Function. 214:181-199.
Rocher, A.B., Crimins, J.I., Amatrudo, J.M., Kinson, M.S., Todd-Brown, M.A., Lewis, J., Luebke, J.I. (2010) Structural and functional changes in tau mutant mice neurons are not linked to the presence of NFTs. Experimental Neurology. 223(2):385-93.
Image 1: Representative rhesus monkey brain, prefrontal cortical slice and biocytin-filled layer 2/3 pyramidal cell. A, Lateral view of the rhesus monkey brain. Boxed area indicates area of the PFC from which tissue is obtained. B, Low power photomicrograph of a PFC slice. Recordings are obtained from pyramidal cells in layer 2/3 in the lower bank of sulcus principalis (arrow head) and in layer 5. Scale bar: 2 mm. C, Photomicrograph of a representative biocytin-filled layer 2/3 pyramidal cell from a 21 year old monkey. Scale bar: 30 µm.
Image 2: One of the electrophysiology rigs that we employ to obtain data is shown in the left panel. This rig is comprised, in part, of a Nikon IR-DIC microscope upon the stage of which is the perfusion chamber which contains an in vitro slice of the prefrontal cortex (upper right panel). The lower right panel shows 2 visually identified layer 2/3 pyramidal cells from which recordings were obtained.
Image 3: Confocal images of representative streptavidin-Alexa 546-labeled layer 3 pyramidal cells from WT (left) and TG (center and right) mice. Top row: 10x confocal images of the cells with Thioflavin-S staining showing a high density of NFTs in the cortex of TG mice. Middle row: Somata of cells shown in top row at 40x. Imaging the Thioflavin-S staining in the soma allows for the classification of the TG cells into one of 2 groups based on the presence or absence of a NFT, respectively named NFT- cells (center column) and NFT+ cells (right column). Bottom row: 3-D reconstruction of cells imaged at high resolution (xy and xz projections on the left and right side of each panel, respectively). Scale bars: top row = 40 µm; middle row = 5 µm.
Image 4:Comparison of electrophysiological properties of prefrontal versus visual cortical pyramidal cells. A) Low magnification photomicrograph of in vitro slices of dlPFC (left) and V1 (right) under IR-DIC optics. Note the significantly smaller neuronal somata in V1 compared to dlPFC. B) Repetitive AP firing in representative dlPFC (red trace) and V1 (blue trace) pyramidal cells in response to +80 pA current step. C) Exemplar traces of sEPSCs from representative dlPFC and V1 cells. D) Left: Averaged traces of all sEPSCs in the dlPFC and V1 cells. Right: Superimposed averaged traces from the dlPFC and V1 cells; V1 response normalized to the peak of the dlPFC response.