Processing of auditory midbrain interspike intervals by model neurons.

A question central to sensory processing is how signal information is encoded and processed by single neurons. Stimulus features can be represented through rate coding (via firing rate), temporal coding (via firing synchronization to temporal periodicities), or temporal encoding (via intricate patterns of spike trains). Of the three, examples of temporal encoding are the least documented. One region in which temporal encoding is currently being explored is the auditory midbrain. Midbrain neurons in the plainfin midshipman generate different interspike interval (ISI) distributions depending on the frequencies of the concurrent vocal signals. However, these distributions differ only along certain lengths of ISIs, so that any neurons trying to distinguish the distributions would have to respond selectively to specific ISI ranges. We used this empirical observation as a realistic challenge with which to explore the plausibility of ISI-tuned neurons that could validate this form of temporal encoding. The resulting modeled cells--point neurons optimized through multidimensional searching--were successfully tuned to discriminate patterns in specific ranges of ISIs. Achieving this task, particularly with simplified neurons, strengthens the credibility of ISI coding in the brain and lends credence to its role in auditory processing.

Tools for physiology labs: an inexpensive high-performance amplifier and electrode for extracellular recording.

The cost of electronic equipment can be a critical barrier to including neurophysiology exercises in biology teaching programs. We describe the construction of a simple and inexpensive AC preamplifier with performance comparable to that of commercial products. The amplifier consists of two integrated circuits in five stages: differential input, fixed gain, variable gain (100 or 1000), low-pass filter (5 or 20 kHz), and 50 or 60 Hz notch filter. We compared our amplifier with two commercial units, the A-M Systems Model 1700 and the Grass P15. The quality of extracellular recording from a typical student preparation (spontaneously active crayfish motor nerve) was the same for all three amplifiers, although our amplifier has slightly higher internal noise than the P15 and slightly lower common-mode rejection than the 1700 and P15. In addition, we describe a simple suction electrode for extracellular nerve recording. It is easily constructed from readily available materials and uses a disposable plastic pipette tip, instead of the traditional glass tip, to contact the nerve. This tip is easily replaced if broken or clogged, and can be adapted to different recording conditions by selecting a different tip size or stretching the plastic. Development of this equipment is part of an ongoing project to promote neuroscience education by expanding the neurophysiology options available to laboratory instructors.

Temporal population code of concurrent vocal signals in the auditory midbrain.

Unique patterns of spike activity across neuron populations have been implicated in the coding of complex sensory stimuli. Delineating the patterns of neural activity in response to varying stimulus parameters and their relationships to the tuning characteristics of individual neurons is essential to ascertaining the nature of population coding within the brain. Here, we address these points in the midbrain coding of concurrent vocal signals of a sound-producing fish, the plainfin midshipman. Midshipman produce multiharmonic vocalizations which frequently overlap to produce beats. We used multivariate statistical analysis from single-unit recordings across multiple animals to assess the presence of a temporal population code. Our results show that distinct patterns of temporal activity emerge among midbrain neurons in response to concurrent signals that vary in their difference frequency. These patterns can serve to code beat difference frequencies. The patterns directly result from the differential temporal coding of difference frequency by individual neurons. Difference frequency encoding, based on temporal patterns of activity, could permit the segregation of concurrent vocal signals on time scales shorter than codes requiring averaging. Given the ubiquity across vertebrates of auditory midbrain tuning to the temporal structure of acoustic signals, a similar temporal population code is likely present in other species.

Monte Carlo simulation of miniature endplate current generation in the vertebrate neuromuscular junction.

A Monte Carlo method for modeling the neuromuscular junction is described in which the three-dimensional structure of the synapse can be specified. Complexities can be introduced into the acetylcholine kinetic model used with only a small increase in computing time. The Monte Carlo technique is shown to be superior to differential equation modeling methods (although less accurate) if a three-dimensional representation of synaptic geometry is desired. The conceptual development of the model is presented and the accuracy estimated. The consequences of manipulations such as varying the spacing of secondary synaptic folds or that between the release of multiple quantal packets of acetylcholine, are also presented. Increasing the spacing between folds increases peak current. Decreased spacing of adjacent quantal release sites increases the potentiation of peak current.

Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.

In previous papers we studied the rising phase of a miniature endplate current (MEPC) to derive diffusion and forward rate constants controlling acetylcholine (AcCho) in the intact neuromuscular junction. The present study derives similar values (but with smaller error ranges) for these constants by including experimental results from the falling phase of the MEPC. We find diffusion to be 4 X 10(-6) cm2 s-1, slightly slower than free diffusion, forward binding to be 3.3 X 10(7) M-1 s-1, and the distance from an average release site to the nearest exit from the cleft to be 1.6 micron. We also estimate the back reaction rates. From our values we can accurately describe the shape of MEPCs under different conditions of receptor and esterase concentration. Since we suggest that unbinding is slower than isomerization, we further predict that there should be several short "closing flickers" during the total open time for an AcCho-ligated receptor channel.

Kinetic parameters for acetylcholine interaction in intact neuromuscular junction.

The dependency of miniature endplate current (mepc) rise time upon mepc amplitude and acetylcholine receptor site density was measured in lizard intercostal muscles and used to fit the rate constants in a simple kinetic scheme. The kinetic scheme included diffusion, two-step sequential binding of acetylcholine to receptor, and opening of the ion channel. Numerical simulation of the observed mepc behavior yielded the following kinetic constants; (i) diffusion constant, 4 X 10(-6) cm2 sec-1; (ii) forward binding rates, 4.7 X 10(7) M-1 sec-1; (iii) channel relaxation rate, 25 msec-1. The value above for the forward binding rates assumed both rates to be equal. If they are different, the slower of the two is in the range of 2-5 X 10(7) M-1 sec-1. A radial profile of bound receptor indicated that activation of the receptor was very local, occurring essentially within a radius of about 0.3 micrometers from the point of acetylcholine release.

Acetylcholine receptor site density affects the rising phase of miniature endplate currents.

The relationship between acetylcholine receptor (AcChoR) site density (sigma) and the rising phase of the miniature endplate current was determined in esterase-inactivated lizard intercostal neuromuscular junctions. The currents were recorded by using a voltage clamp. The receptor site density was determined by electron microscope autoradiography after labeling with 125I-labeled alpha-bungarotoxin in normal endplates and in those partially inactivated with nonradioactive alpha-bungarotoxin. We found that as sigma is decreased the rise time in increased and the amplitude is decreased. These results are compatible with a previously stated "saturating disk" model, which suggests that a quantum of acetylcholine (AcCho) acts on a small postsynaptic area at saturating concentration. We conclude that in the normal neuromuscular junction the most likely number of AcCho molecules needed to open an ion channel is 2, and that the 20--80% rise time of < 100 musec is influenced both by the sigma-dependent factors such as diffusion and binding of AcCho to AcChoR and by the sigma-independent time delays such as the conformation change time to open the ion channels. From our data we calculate the lower limits to the forward rate constant of AcCho binding to AcChoR greater than or equal to 3 X 10(7) M-1 sec-1 and the diffusion constant for AcCho in the cleft greater than or equal to 4 X 10(-6) cm2 sec-1.

Acetylcholine receptor distribution on myotubes in culture correlated to acetylcholine sensitivity.

1. A linear relation, with a slope of 0-9 +/- 0-2 on a log-log plot, was obtained between acetylcholine (ACh) sensitivity and alpha-bungarotoxin (alpha-BTX) binding site density in developing L6 and rat primary myotubes. ACh sensitivity was defined as g/Qn where g is conductance, Q is ACh charge and n is the Hill coefficient. Experimentally we found n approximately 1-7 for our myotubes, which is similar in value to that reported for adult systems. 2. The linear relationship is compatible with an organization whereby each ion channel is always complexed with a fixed number of ACh receptors such that the dose-response characteristics of each such complex are independent of average ACh receptor density. 3. Light microscope autoradiography showed that the alpha-bungarotoxin binding sites on L6 myotubes are uniformly distributed over the surface, while primary rat myotubes exhibit gradients and hot spots. Electron microscope autoradiography indicated that about 70% of the [125I]alpha-bungarotoxin label was on the surface of the myotubes. The alpha-bungarotoxin site density, after subtracting myoblast background, varied from 5 to 400 sites/micrometer2 on different L6 myotubes, and from 54 to 900 sites/micrometer2 on primary rat myotubes, with occasional hot spots of 3000-4000 sites/micrometer2. The conductance sensitivities varied from 10(-4) to 2 X 10(-2) Momega-1/nC1-7.