Using the Tools of Behavioral Neuroscience to Determine the Identity of Different Mouse Strains in a Laboratory Course.

Understanding the neural mechanisms underlying behavior depends on our ability to define and to measure these behaviors in the model animal. We describe an upper-level course which provides students with hands-on experience in the methods of behavioral neuroscience. There are many well-established behavioral tests which are relatively easy for students to conduct that can be used to determine the performance of animals in such tasks as anxiety, motor performance and memory. Laboratory mice bred specifically to exhibit particular behavioral characteristics are readily available from vendors along with well documented behavioral profiles for these strains. We used two albino strains CD1 and BALBc as our model animals. Students were given the task of identifying the strains based on the results of a battery of behavioral tests but were not given information about the mice. These two strains were chosen for their clear differences particularly in tests of anxiety. Students conducted elevated plus maze and zero maze tests, open field test, light-dark exploratory task, rotarod, balance beam test, spatial or novel object learning. Students were able to correctly identify the two strains by comparing their own data with the published literature in the field. The course structure encouraged students to work in teams to design protocols, and then to collect and explore data. Students were enthusiastic about the hands-on laboratory experience and were able to demonstrate an appreciation for and understanding of these methods in behavioral neuroscience.

Studying temporal properties of stimulus-evoked responses in the ventral nerve cord of insects.

Students in undergraduate laboratory settings learn many of the foundational principles of sensory processing in the comparatively simple, easy to study invertebrate nervous system. In this example preparation, the American cockroach, students record action potentials from the fibers in the ventral nerve cord (VNC) that participate in a well explained escape behavior in response to stimulation of its cerci, a pair of mechanosensitive abdominal appendages. A system that allows good control over the time and amplitude of the air pulse delivered to the cerci is described. This experimental setup enables students to extract and display temporal information from recordings to learn how to interpret those responses in the context of the properties of the stimulus. I offer examples of specific investigations and analyses that work well for this purpose in an undergraduate laboratory.

The multiple functions of T stellate/multipolar/chopper cells in the ventral cochlear nucleus.

Acoustic information is brought to the brain by auditory nerve fibers, all of which terminate in the cochlear nuclei, and is passed up the auditory pathway through the principal cells of the cochlear nuclei. A population of neurons variously known as T stellate, type I multipolar, planar multipolar, or chopper cells forms one of the major ascending auditory pathways through the brainstem. T Stellate cells are sharply tuned; as a population they encode the spectrum of sounds. In these neurons, phasic excitation from the auditory nerve is made more tonic by feedforward excitation, coactivation of inhibitory with excitatory inputs, relatively large excitatory currents through NMDA receptors, and relatively little synaptic depression. The mechanisms that make firing tonic also obscure the fine structure of sounds that is represented in the excitatory inputs from the auditory nerve and account for the characteristic chopping response patterns with which T stellate cells respond to tones. In contrast with other principal cells of the ventral cochlear nucleus (VCN), T stellate cells lack a low-voltage-activated potassium conductance and are therefore sensitive to small, steady, neuromodulating currents. The presence of cholinergic, serotonergic and noradrenergic receptors allows the excitability of these cells to be modulated by medial olivocochlear efferent neurons and by neuronal circuits associated with arousal. T Stellate cells deliver acoustic information to the ipsilateral dorsal cochlear nucleus (DCN), ventral nucleus of the trapezoid body (VNTB), periolivary regions around the lateral superior olivary nucleus (LSO), and to the contralateral ventral lemniscal nuclei (VNLL) and inferior colliculus (IC). It is likely that T stellate cells participate in feedback loops through both medial and lateral olivocochlear efferent neurons and they may be a source of ipsilateral excitation of the LSO.

Ca2+-dependent, stimulus-specific modulation of the plasma membrane Ca2+ pump in hippocampal neurons.

The plasma membrane Ca(2+) ATPase (PMCA) plays a major role in restoring Ca(2+) to basal levels following transient elevation by neuronal activity. Here we examined the effects of various stimuli that increase [Ca(2+)](i) on PMCA-mediated Ca(2+) clearance from hippocampal neurons. We used indo-1-based microfluorimetry in the presence of cyclopiazonic acid to study the rate of PMCA-mediated recovery of Ca(2+) elevated by a brief train of action potentials. [Ca(2+)](i) recovery was described by an exponential decay and the time constant provided an index of PMCA-mediated Ca(2+) clearance. PMCA function was assessed before and for >or=60 min following a 10-min priming stimulus of either 100 microM N-methyl-d-aspartate (NMDA), 0.1 mM Mg(2+) (reduced extracellular Mg(2+) induces intense excitatory synaptic activity), 30 mM K(+), or control buffer. Recovery kinetics slowed progressively following priming with NMDA or 0.1 mM Mg(2+); in contrast, Ca(2+) clearance initially accelerated and then slowly returned to initial rates following priming with 30 mM K(+)-induced depolarization. Treatment with 10 muM calpeptin, an inhibitor of the Ca(2+) activated protease calpain, prevented the slowing of kinetics observed following treatment with NMDA but had no affect on the recovery kinetics of control cells. Calpeptin also blocked the rapid acceleration of Ca(2+) clearance following depolarization. In calpeptin-treated cells, 0.1 mM Mg(2+) induced a graded acceleration of Ca(2+) clearance. Thus in spite of producing comparable increases in [Ca(2+)](i), activation of NMDA receptors, depolarization-induced activation of voltage-gated Ca(2+) channels and excitatory synaptic activity each uniquely affected Ca(2+) clearance kinetics mediated by the PMCA.

Responses to sounds in the central auditory system of the frog: an advanced electrophysiology laboratory in sensory processing.

Frogs rely upon vocal communication to advertise for potential mates, to defend territory and to alarm neighbors of danger. Cells in the auditory midbrain of an awake frog display tuning to the spectral energy present in calls based upon discharge rate and encode the temporal properties of calls in the timing of their discharges. This laboratory experiment is designed to allow students to explore the relationship between stimulus amplitude or frequency and response rate, and how the timing of responses can also be used to encode behaviorally relevant features of the stimulus. Action potentials in the midbrain auditory nucleus, the torus semicularis, are evoked by delivery of free field sounds and recorded. Most cells are broadly tuned to frequency, yet some can be fairly precise in preserving periodic structure. The use of a comparative model of study should help students understand principles common among all sensory systems, and an appreciation that the architecture of each system is adaptively matched to the ethological task at hand.

The emergence of temporal hyperacuity from widely tuned cell populations.

Typically, individual neural cells operate on a millisecond time scale yet behaviorally animals reveal sub-microsecond acuity. Our model resolves this huge discrepancy by using populations of many widely tuned cells to attain sub-microsecond resolution in a temporal discrimination task. An echolocating bat uses its auditory system to locate objects and it demonstrates remarkable temporal precision in psychophysical tasks. Auditory cells were simulated using realistic parameters and connected in three ascending layers with descending projections from auditory cortex. Coincidence detection of firing collicular cells at thalamus and subsequent integration of multiple inputs at cortex, produce an estimate of time represented as the mean of the active cortical population. Multiple estimates allow the model bat to use memory to recognize predictable change in stimuli values. The best performance is produced using cortical feedback and a computation of target time based on combining the current and previous estimates. Temporal hyperacuity is attained through population coding of physiologically realistic cells but depends on the inherent properties of the psychophysical task.

Delay accuracy in bat sonar is related to the reciprocal of normalized echo bandwidth, or Q.

Big brown bats (Eptesicus fuscus) emit wideband, frequency-modulated biosonar sounds and perceive the distance to objects from the delay of echoes. Bats remember delays and patterns of delay from one broadcast to the next, and they may rely on delays to perceive target scenes. While emitting a series of broadcasts, they can detect very small changes in delay based on their estimates of delay for successive echoes, which are derived from an auditory time/frequency representation of frequency-modulated sounds. To understand how bats perceive objects, we need to know how information distributed across the time/frequency surface is brought together to estimate delay. To assess this transformation, we measured how alteration of the frequency content of echoes affects the sharpness of the bat's delay estimates from the distribution of errors in a psychophysical task for detecting changes in delay. For unrestricted echo frequency content and high echo signal-to-noise ratio, bats can detect extremely small changes in delay of about 10 ns. When echo bandwidth is restricted by filtering out low or high frequencies, the bat's delay acuity declines in relation to the reciprocal of relative echo bandwidth, expressed as Q, which also is the relative width of the target impulse response in cycles rather than time. This normalized-time dimension may be efficient for target classification if it leads to target shape being displayed independent of size. This relation may originate from cochlear transduction by parallel frequency channels with active amplification, which creates the auditory time/frequency representation itself.

Transformation of external-ear spectral cues into perceived delays by the big brown bat, Eptesicus fuscus.

The external-ear transfer function for big brown bats (Eptesicus fuscus) contains two prominent notches that vary from 30 to 55 kHz and from 70 to 100 kHz, respectively, as sound-source elevation moves from -40 to +10 degrees. These notches resemble a higher-frequency version of external-ear cues for vertical localization in humans and other mammals. However, they also resemble interference notches created in echoes when reflected sounds overlap at short time separations of 30-50 micros. Psychophysical experiments have shown that bats actually perceive small time separations from interference notches, and here we used the same technique to test whether external-ear notches are recognized as a corresponding time separation, too. The bats' performance reveals the elevation dependence of a time-separation estimate at 25-45 micros in perceived delay. Convergence of target-shape and external-ear cues onto echo spectra creates ambiguity about whether a particular notch relates to the object or to its location, which the bat could resolve by ignoring the presence of notches at external-ear frequencies. Instead, the bat registers the frequencies of notches caused by the external ear along with notches caused by the target's structure and employs spectrogram correlation and transformation (SCAT) to convert them all into a family of delay estimates that includes elevation.

Octopus cells of the mammalian ventral cochlear nucleus sense the rate of depolarization.

Whole cell patch recordings in slices show that the probability of firing of action potentials in octopus cells of the ventral cochlear nucleus depends on the dynamic properties of depolarization. Octopus cells fired only when the rate of rise of a depolarization exceeded a threshold value that varied between 5 and 15 mV/ms among cells. The threshold rate of rise was independent of whether depolarizations were evoked synaptically or by the intracellular injection of current. Previous work showed that octopus cells are contacted by many auditory nerve fibers, each providing less than 1-mV depolarization. Summation of synaptic input from multiple fibers is required for an octopus cell to reach threshold. In firing only when synaptic depolarization exceeds a threshold rate, octopus cells fire selectively when synaptic input is sufficiently large and synchronized for the small, brief unitary excitatory postsynaptic potentials (EPSPs) to sum to produce a rapidly rising depolarization. The sensitivity to rate of depolarization is governed by a low-threshold, alpha-dendrotoxin-sensitive potassium conductance (g(KL)). This conductance also shapes the peaks of action potentials, contributing to the precision in their timing. Firing in neighboring T stellate cells depends much less strongly on the rate of rise. They lack strong alpha-dendrotoxin-sensitive conductances. Octopus cells appear to be specialized to detect synchronization in the activation of groups of auditory nerve fibers, a common pattern in responses to natural sounds, and convey its occurrence with temporal precision.