- Anatomy, Physiology, and Synaptic Responses of Rat Layer V Auditory Cortical Cells and Effects of Intracellular GABAA Blockade

The methods described here for intracellular sharp microelectrode recording are similar to those described previously (Smith1992ref-arrow.gif). All methods were approved by the University of WisconsinAnimal Care and Use Committee. Animals were maintained in an AmericanAssociations for Accreditation of Laboratory Animal Care (AAALAC)-approvedfacility. Three to 6-wk-old Long-Evans hooded rats were givenan anesthetic overdose of chloral hydrate solution (70 mg/ml ip).When areflexive, rats were perfused transcardially with cold,oxygenated sucrose saline (described at end of paragraph). Thebrain was then exposed dorsally, and cuts in the coronal planewere made halfway through the rostrocaudal extent of the cerebellumand one-third of the way through the rostrocaudal extent of thecerebral cortex. The block of tissue between these two cuts wasremoved and glued either ventral side down (for horizontal sections)or rostral side down (for coronal sections). The tissue was thensubmerged in cold, oxygenated saline, and 400- to 500-µm sectionswere cut through primary auditory cortex (Te1) on a vibratome.To preserve more of the axonal projection from the medial geniculatebody (MGB) to Te1 in horizontal slices, the tissue was blockedsomewhat higher rostrally and laterally with wedges of fixed eggalbumin (Metherate 1999ref-arrow.gif). Sections containing Te1 were placedin a holding chamber containing normal, oxygenated artificialcerebrospinal fluid (ACSF) at room temperature. After equilibratingin the holding chamber for at least 15 min, one slice was transferredto the recording chamber, where it was placed between two piecesof nylon mesh and perfused with normal, oxygenated ACSF at 35°C,which contained the following (in mM): 124 NaCl, 5 KCl, 1.2 KH2PO4,2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3, and 10 glucose. The sucrose salinecontained sucrose in place of NaCl (Aghajanian and Rasmussen 1989ref-arrow.gif).The slice was then allowed to rest a minimum of 45 additionalminutes before recordingbegan.

Bipolar stimulating electrodes were used to activate axons in the white matter in coronal slices, with stimuli that were steppedfrom 10 to 150 V in 10-V increments, and with durations of 100 or 200 µs. In horizontal slices, the internal capsule and externalcapsule were stimulated separately, allowing isolation of thalamocorticalfrom corticocortical inputs (Fig. 1). The space between the pairedelectrode tips was sufficient to span the width of the fiber tractto stimulate the maximum number of inputs possible. Occasionally,stimulation induced antidromic spikes from the recorded cell.If this was observed, the polarity of the electrode was switchedor the stimulating electrode was moved.

Intracellular recordings of responses to injected current and evoked postsynaptic potentials were made with glass microelectrodesof 70-150 MOmega resistance when filled with 2 M potassium acetateand 2% Neurobiotin (Vector Laboratories, Burlingame, CA). Onlycells with resting potentials more negative than -60 mV and overshootingaction potentials were used for statistical analysis and illustration.Intracellular current and voltage records were digitized withcustom software (ICEPAC, L. Haberly, University of Wisconsin).A neuron's membrane potential was calculated by subtracting thecell's recorded voltage from the extracellular DC potential justafter exiting the cell. The input resistance was calculated usingthe slope of the linear portion of the current-voltage plot nearthe cell's resting potential. Voltage was averaged over 100 msduring the last 120 ms of a 300- or 400-ms current pulse. Duringrecording, Neurobiotin was injected into the cell for ~5 min with0.4- to 0.6-nA current pulses. To quantify spike half-widths,the first and fifth spikes were measured. The fifth spike waschosen because it was usually the first or second spike afterthe burst in IB cells, and all cells fired at least five spikesin response to current injection. Measurements were taken at thelowest current injection strength at which the cell fired fivespikes, which was always between 0.1 and 0.5 nA. Synaptic latencieswere measured from the center of the stimulus artifact, whichwas usually a total of 0.5 ms in duration, to the onset of thevoltage deflection. Inhibitory postsynaptic potential (IPSP) latencywas measured from the center of the stimulus artifact to the onsetof the IPSP, which was identified as the onset of the change inthe slope of the voltage deflection that reversed at levels correspondingto a chloride or mixed anion conductance (usually between -50and -70mV).

GABAB was blocked with saclofen (Research Biochemicals International, Natick, MA) in three cells, one IB and two RS. GABAA-activatedchloride channels were blocked intracellularly with 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxi-calix[4]arene(TS-TM calix[4]arene) and 5,11,17,23-tetrasulfonato-calix[4]arene(TS calix[4]arene), which were generously provided by Dr. AshvaniSingh at the University of Pittsburgh. These compounds were usedat a concentration of 1-5 µM. They were injected into the cellafter control trials were taken using hyperpolarizing square currentpulses (300 ms current pulse every 800 ms). It usually took between20 and 40 min for the chloride blockers to takeeffect.

After recording was complete, the slice was fixed in fresh 4% paraformaldehyde. It was then cryoprotected, and 60-µm frozensections were cut on a freezing microtome and collected in 0.1 M phosphate buffer, pH 7.4. The sections were incubated in avidin-biotin-HRPcomplex (ABC kit, Vector Labs). The following day, they were rinsedin phosphate buffer and incubated with nickel/cobalt-intensifieddiaminobenzidine (DAB) (Adams 1981ref-arrow.gif). The sections were then mounted,counterstained with cresyl violet, andcoverslipped.

Drawings of injected cells were made using a camera lucida attached to a Zeiss microscope. The location of the cell body relativeto the areas of rat cerebral cortex was determined using the atlasof Paxinos and Watson (1986)ref-arrow.gif and studies in which evoked potentialrecordings were used to map the location of primary auditory cortex(Barth and Di 1990ref-arrow.gif, 1991ref-arrow.gif; Di and Barth 1992ref-arrow.gif). Cells were determinedto be within layer V by two means. The first was inspection ofindividual sections, where differences in cell size, density andshape were used to indicate transitions between cortical layers.The second means to determine laminar borders in Te1 was to usepreviously established measures of layer V laminar borders (Gamesand Winer 1988ref-arrow.gif), in which layer V is defined as the region ~51-77%of the distance through Te1 when measured from the pial surface.Only those cells that fell both within primary auditory cortex(Te1) and layer V were used for analysis. Anatomic measurementswere made using a Neurolucida drawing system (MicroBrightField,Colchester, VT). All statistical analyses were done using Minitab(Minitab, State College, PA). Depending on the data set, eithertwo-sided two-sample t-tests or chi2 tests wereused.


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