Lab Features

Slice-patch electrophysiology recording	This system allows intracellular recording from individual cells either with a sharp microelectrode or with a patch electrode. With the perforated patch technique we are currently using nystatin to gain access to the cell interior. Recordings examine membrane properties, action potential characteristics, measurements of conductance through current -voltage relationships (I-V), excitatory and inhibitory synaptic events. We are using these cellular techniques to study the effects of specific neurotransmitters and neuropeptides within the brainstem parabrachial nucleus, and the diagonal band of Broca (DBB), a forebrain nucleus.
Dissociated cell electrophysiology recording	Whole cell patch clamp recordings from acutely dissociated neurons permit a high resolution analysis of the ionic mechanisms underlying the actions of transmitters in the CNS. We have exploited this technique to record from DBB neurons and assess the mechanisms of actions of a wide variety of chemical messengers involved in neurotransmission in the forebrain. This technique can be combined with Single-cell RT-PCR analysis to determine the chemical identity of neurons under study or the presence of specific channel subtypes on such neurons. We are in the process of establishing a primary culture of basal forebrain neurons that will permit us to examine issues related to cell survival or death following exposure to amyloid peptides
DIC Slice patch recording	DIC slice patch recording is similar to blind slice patch recording except that you are able to visualize the first few layers of cells and therefore can, under visual guidance, approach and patch a particular cell that you have observed as being healthy. In addition, we are able to use Cy3 192 IgG saporin to label cholinergic cells within the diagonal band of Broca which project to the hippocampus. These cells are then detectable with a fluorescent light source. We use a Ziess Axioskop II fs for our slice patch recording.
Single channel recording	We use electrophysiological patch clamp techniques to record isolated ion currents from acutely dissociated diagonal band of Broca (DBB) neurons and cultured basal forebrain neurons. Cell-attached and outside-out single-channel patch-clamp techniques are used in exploiting non-a7 nicotinic acetylcholine receptor (nAChR) functions. We are particularly interested in the mechanisms of nAChRs and interaction with beta amyloid, neurotransmitters, and drugs related to AD and neurodegeneration diseases. We quantitatively analyze single channel activity and combined with single-cell RT-PCR and Western blot techniques to identify the specified subtypes and mechanisms of nAChRs. Future work will use electrophysiology coupled with molecular biological approaches the nAChR ionic mechanisms and related signal transduction pathways.
Primary basal forebrain neuronal culture
Calcium imaging	Intracellular Ca²⁺ regulates and modulates many neuronal processes such as release of neurotransmitters, gene expression, and apoptotic pathways. Using our imaging system, we can examine how various drugs influence intracellular Ca²⁺ levels in neurons. In order to measure intracellular Ca²⁺, neurons are loaded with Fura-2, a Ca²⁺ sensitive fluorescent dye. We excite the intracellular Fura-2 with specific wavelengths of ultraviolet light emitted by a monochromater. The intensity of the fluorescence emitted by Fura-2 is measured with a digital camera and used to calculate the intracellular Ca²⁺ concentration.
Immunohistochemestry, in situ hybridization, and single cell RT-PCR.	Rat blood pressure is altered by the use of peptides or drugs so we may better understand the compensation effects that the rat brain takes when challenged. To study this effect we use a combination of Immunohistochemistry ( to look for activated neurons) and in situ hybridization (to look for change in message mRNA) in the rat brain. Some of the messages we are looking for are Neuropeptide Y, Neuropeptide FF, and Corticotropin Releasing Factor.

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