Spike detection, characterization, and discrimination using feature analysis software written in LabVIEW.

Rapid and accurate discrimination of single units from extracellular recordings is a fundamental process for the analysis and interpretation of electrophysiological recordings. We present an algorithm that performs detection, characterization, discrimination, and analysis of action potentials from extracellular recording sessions. The program was entirely written in LabVIEW (National Instruments), and requires no external hardware devices or a priori information about action potential shapes. Waveform events are detected by scanning the digital record for voltages that exceed a user-adjustable trigger. Detected events are characterized to determine nine different time and voltage levels for each event. Various algebraic combinations of these waveform features are used as axis choices for 2-D Cartesian plots of events. The user selects axis choices that generate distinct clusters. Multiple clusters may be defined as action potentials by manually generating boundaries of arbitrary shape. Events defined as action potentials are validated by visual inspection of overlain waveforms. Stimulus-response relationships may be identified by selecting any recorded channel for comparison to continuous and average cycle histograms of binned unit data. The algorithm includes novel aspects of feature analysis and acquisition, including higher acquisition rates for electrophysiological data compared to other channels. The program confirms that electrophysiological data may be discriminated with high-speed and efficiency using algebraic combinations of waveform features derived from high-speed digital records.

Cerebellar unipolar brush cells are targets of primary vestibular afferents: an experimental study in the gerbil.

The unipolar brush cell (UBC) is an excitatory glutamatergic interneuron, situated in the cerebellar granular layer, that itself receives excitatory synaptic input on its dendritic brush from a single mossy fiber terminal in the form of a giant glutamatergic synapse. The UBC axon branches within the granular layer, giving rise to large terminals that synapse with both granule cell and UBC dendrites within glomeruli and resemble in morphological and functional terms those formed by extrinsic mossy fibers. So far, the only demonstrated extrinsic afferents to the UBC are the choline acetyltransferase (ChAT)-positive mossy fibers, some of which originate from the medial and descending vestibular nuclei. To ascertain whether UBCs are innervated by primary vestibular fibers, we performed a tract-tracing light and electron microscopic study of the vestibulocerebellum in gerbils. Macular and canal vestibular end-organs were individually labeled by injection of biotinylated dextran amine. After an appropriate survival time, gerbils were then processed for light and electron microscopic analysis of central vestibular projections. In the nodulus and uvula, labeled primary vestibular fibers formed mossy terminals synapsing with both granule cells and UBCs in all of the injected gerbils. Thus, innervation of UBCs by extrinsic mossy fibers carrying static and dynamic vestibular signals represents the first synapse of networks that contribute a powerful form of distributed excitation in the granular layer.

Peripheral patterns of terminal innervation of vestibular primary afferent neurons projecting to the vestibulocerebellum in the gerbil.

Retrograde transganglionic labeling techniques with biotinylated dextran amine (BDA) were used to examine the terminal field structure and topographical patterns of innervation within the vestibular sensory end organs of vestibular primary afferent neurons projecting to the cerebellar uvula/nodulus and flocculus lobules in the gerbil. Robust, dark labeling in the cristae ampullares suggested that the vast majority of the terminals of afferent neurons were of the dimorphic type. The majority (94% to the uvula/nodulus and 100% to the flocculus) innervates the peripheral zones of each of the three semicircular canal cristae. Comparison of the type and distribution of terminals across the canalicular sensory neuroepithelium with morphophysiological studies in chinchilla suggests that the labeled population consists predominantly of peripheral terminal fields of lower-to-intermediate gain, more regularly firing, tonic afferents. For otolith organ-related afferents, the uvula/nodulus receives strong inputs from primary otolith afferent neurons identified as dimorphic in type that predominately innervate the peristriolar zones of the utricular and saccular maculae. No direct otolith organ-related inputs to the flocculus were observed. In contrast to the canal afferents, the types and locations of labeled otolith afferent terminals suggest that they largely consist of irregularly firing, high-gain, phasic neurons.

Vestibular efferent neurons project to the flocculus.

A bilateral projection from the vestibular efferent neurons, located dorsal to the genu of the facial nerve, to the cerebellar flocculus and ventral paraflocculus was demonstrated. Efferent neurons were double-labeled by the unilateral injections of separate retrograde tracers into the labyrinth and into the floccular and ventral parafloccular lobules. Efferent neurons were found with double retrograde tracer labeling both ipsilateral and contralateral to the sites of injection. No double labeling was found when using a fluorescent tracer with non-fluorescent tracers such as horseradish peroxidase (HRP) or biotinylated dextran amine (BDA), but large percentages of efferent neurons were found to be double labeled when using two fluorescent substances including: fluorogold, microruby dextran amine, or rhodamine labeled latex beads. These data suggest a potential role for vestibular efferent neurons in modulating the dynamics of the vestibulo-ocular reflex (VOR) during normal and adaptive conditions.

Convergent properties of vestibular-related brain stem neurons in the gerbil.

Three classes of vestibular-related neurons were found in and near the prepositus and medial vestibular nuclei of alert or decerebrate gerbils, those responding to: horizontal translational motion, horizontal head rotation, or both. Their distribution ratios were 1:2:2, respectively. Many cells responsive to translational motion exhibited spatiotemporal characteristics with both response gain and phase varying as a function of the stimulus vector angle. Rotationally sensitive neurons were distributed as Type I, II, or III responses (sensitive to ipsilateral, contralateral, or both directions, respectively) in the ratios of 4:6:1. Four tested factors shaped the response dynamics of the sampled neurons: canal-otolith convergence, oculomotor-related activity, rotational Type (I or II), and the phase of the maximum response. Type I nonconvergent cells displayed increasing gains with increasing rotational stimulus frequency (0.1-2.0 Hz, 60 degrees /s), whereas Type II neurons with convergent inputs had response gains that markedly decreased with increasing translational stimulus frequency (0.25-2.0 Hz, +/-0.1 g). Type I convergent and Type II nonconvergent neurons exhibited essentially flat gains across the stimulus frequency range. Oculomotor-related activity was noted in 30% of the cells across all functional types, appearing as burst/pause discharge patterns related to the fast phase of nystagmus during head rotation. Oculomotor-related activity was correlated with enhanced dynamic range compared with the same category that had no oculomotor-related response. Finally, responses that were in-phase with head velocity during rotation exhibited greater gains with stimulus frequency increments than neurons with out-of-phase responses. In contrast, for translational motion, neurons out of phase with head acceleration exhibited low-pass characteristics, whereas in-phase neurons did not. Data from decerebrate preparations revealed that although similar response types could be detected, the sampled cells generally had lower background discharge rates, on average one-third lower response gains, and convergent properties that differed from those found in the alert animals. On the basis of the dynamic response of identified cell types, we propose a pair of models in which inhibitory input from vestibular-related neurons converges on oculomotor neurons with excitatory inputs from the vestibular nuclei. Simple signal convergence and combinations of different types of vestibular labyrinth information can enrich the dynamic characteristics of the rotational and translational vestibuloocular responses.

Primate translational vestibuloocular reflexes. III. Effects of bilateral labyrinthine electrical stimulation.

The effects of functional, reversible ablation and potential recruitment of the most irregular otolith afferents on the dynamics and sensitivity of the translational vestibuloocular reflexes (trVORs) were investigated in rhesus monkeys trained to fixate near and far targets. Translational motion stimuli consisted of either steady-state lateral and fore-aft sinusoidal oscillations or short-lasting transient lateral head displacements. Short-duration (usually <2 s) anodal (inhibitory) and cathodal (excitatory) currents (50-100 microA) were delivered bilaterally during motion. In the presence of anodal labyrinthine stimulation, trVOR sensitivity and its dependence on viewing distance were significantly decreased. In addition, anodal currents significantly increased phase lags. During transient motion, anodal stimulation resulted in significantly lower initial eye acceleration and more sluggish responses. Cathodal currents tended to have opposite effects. The main characteristics of these results were simulated by a simple model where both regularly and irregularly discharging afferents contribute to the trVORs. Anodal labyrinthine currents also were found to decrease eye velocity during long-duration, constant velocity rotations, although results were generally more variable compared with those during translational motion.

Correlation of Fos expression and circling asymmetry during gerbil vestibular compensation.

Vestibular compensation is a central nervous system process resulting in recovery of functional movement and control following a unilateral vestibular lesion. Small pressure injections of phosphorothioate 20mer oligonucleotides were used to probe the role of the Fos transcription protein during vestibular compensation in the gerbil brainstem. During isoflurane gas anesthesia, antisense probes against the c-fos mRNA sequence were injected into the medial vestibular and prepositus nuclei unilaterally prior to a unilateral surgical labyrinthectomy. Anionic dyes, which did not interact with the oligonucleotides, were used to mark the injection site and help determine the extent of diffusion. The antiFos oligonucleotide injections reduced Fos expression at the injection site in neurons which normally express Fos after the lesion, and also affected circling behavior induced by hemilabyrinthectomy. With both ipsilateral and contralateral medial vestibular and prepositus nuclei injections, less ipsilateral and more contralateral circling was noted in animals injected with antiFos injections as compared to non-injected controls. The degree of change in these behaviors was dependent upon the side of the injection. Histologically, antiFos injections reduced the number of Fos immunolabeled neurons around the injection site, and increased Fos expression contralaterally. The correlation of the number of neurons with Fos expression to turning behavior was stronger for contralateral versus ipsilateral turns, and for neurons in the caudal and ipsilateral sub-regions of the medial vestibular and prepositus nuclei. The results are discussed in terms of neuronal firing activity versus translational activity based on the asymmetrical expression of the Fos inducible transcription factor in the medial vestibular and prepositus nuclei. Although ubiquitous in the brain, transcription factors like Fos can serve localized and specific roles in sensory-specific adaptive stimuli. Antisense injections can be an effective procedure for localized intervention into complex physiological functions, e.g. vestibular compensation.

Saccule contribution to immediate early gene induction in the gerbil brainstem with posterior canal galvanic or hypergravity stimulation.

Immunolabeling patterns of the immediate early gene-related protein Fos in the gerbil brainstem were studied following stimulation of the sacculus by both hypergravity and galvanic stimulation. Head-restrained, alert animals were exposed to a prolonged (1 h) inertial vector of 2 G (19.6 m/s2) head acceleration directed in a dorso-ventral head axis to maximally stimulate the sacculus. Fos-defined immunoreactivity was quantified, and the results compared to a control group. The hypergravity stimulus produced Fos immunolabeling in the dorsomedial cell column (dmcc) of the inferior olive independently of other subnuclei. Similar dmcc labeling was induced by a 30 min galvanic stimulus of up to -100 microA applied through a stimulating electrode placed unilaterally on the bony labyrinth overlying the posterior canal (PC). The pattern of vestibular afferent firing activity induced by this galvanic stimulus was quantified in anesthetized gerbils by simultaneously recording from Scarpa's ganglion. Only saccular and PC afferent neurons exhibited increases in average firing rates of 200-300%, suggesting a pattern of current spread involving only PC and saccular afferent neurons at this level of stimulation. These results suggest that alteration in saccular afferent firing rates are sufficient to induce Fos-defined genomic activation of the dmcc, and lend further evidence to the existence of a functional vestibulo-olivary-cerebellar pathway of adaptation to novel gravito-inertial environments.

Three-dimensional analysis of vestibular efferent neurons innervating semicircular canals of the gerbil.

Anterograde labeling techniques were used to examine peripheral innervation patterns of vestibular efferent neurons in the crista ampullares of the gerbil. Vestibular efferent neurons were labeled by extracellular injections of biocytin or biotinylated dextran amine into the contralateral or ipsilateral dorsal subgroup of efferent cell bodies (group e) located dorsolateral to the facial nerve genu. Anterogradely labeled efferent terminal field varicosities consist mainly of boutons en passant with fewer of the terminal type. The bouton swellings are located predominately in apposition to the basolateral borders of the afferent calyces and type II hair cells, but several boutons were identified close to the hair cell apical border on both types. Three-dimensional reconstruction and morphological analysis of the terminal fields from these cells located in the sensory neuroepithelium of the anterior, horizontal, and posterior cristae were performed. We show that efferent neurons densely innervate each end organ in widespread terminal fields. Subepithelial bifurcations of parent axons were minimal, with extensive collateralization occurring after the axons penetrated the basement membrane of the neuroepithelium. Axonal branching ranged between the 6th and 27th orders and terminal field collecting area far exceeds that of the peripheral terminals of primary afferent neurons. The terminal fields of the efferent neurons display three morphologically heterogeneous types: central, peripheral, and planum. All cell types possess terminal fields displaying a high degree of anisotropy with orientations typically parallel to or within +/-45 degrees of the longitudinal axis if the crista. Terminal fields of the central and planum zones predominately project medially toward the transverse axis from the more laterally located penetration of the basement membrane by the parent axon. Peripheral zone terminal fields extend predominately toward the planum semilunatum. The innervation areas of efferent terminal fields display a trend from smallest to largest for the central, peripheral, and planum types, respectively. Neurons that innervate the central zone of the crista do not extend into the peripheral or planum regions. Conversely, those neurons with terminal fields in the peripheral or planum regions do not innervate the central zone of the sensory neuroepithelium. The central zone of the crista is innervated preferentially by efferent neurons with cell bodies located in the ipsilateral group e. The peripheral and planum zones of the crista are innervated preferentially by efferent neurons with cell bodies located in the contralateral group e. A model incorporating our anatomic observations is presented describing an ipsilateral closed-loop feedback between ipsilateral efferent neurons and the periphery and an open-loop feed-forward innervation from contralateral efferent neurons. A possible role for the vestibular efferent neurons in the modulation of semicircular canal afferent response dynamics is proposed.

Transneuronal pathways to the vestibulocerebellum.

The alpha-herpes virus (pseudorabies, PRV) was used to observe central nervous system (CNS) pathways associated with the vestibulocerebellar system. Retrograde transneuronal migration of alpha-herpes virions from specific lobules of the gerbil and rat vestibulo-cerebellar cortex was detected immunohistochemically. Using a time series analysis, progression of infection along polyneuronal cerebellar afferent pathways was examined. Pressure injections of > 20 nanoliters of a 10(8) plaque forming units (pfu) per ml solution of virus were sufficient to initiate an infectious locus which resulted in labeled neurons in the inferior olivary subnuclei, vestibular nuclei, and their afferent cell groups in a progressive temporal fashion and in growing complexity with increasing incubation time. We show that climbing fibers and some other cerebellar afferent fibers transported the virus retrogradely from the cerebellum within 24 hours. One to three days after cerebellar infection discrete cell groups were labeled and appropriate laterality within crossed projections was preserved. Subsequent nuclei labeled with PRV after infection of the flocculus/paraflocculus, or nodulus/uvula, included the following: vestibular (e.g., z) and inferior olivary nuclei (e.g., dorsal cap), accessory oculomotor (e.g., Darkschewitsch n.) and accessory optic related nuclei, (e.g., the nucleus of the optic tract, and the medial terminal nucleus); noradrenergic, raphe, and reticular cell groups (e.g., locus coeruleus, dorsal raphe, raphe pontis, and the lateral reticular tract); other vestibulocerebellum sites, the periaqueductal gray, substantia nigra, hippocampus, thalamus and hypothalamus, amygdala, septal nuclei, and the frontal, cingulate, entorhinal, perirhinal, and insular cortices. However, there were differences in the resulting labeling between infection in either region. Double-labeling experiments revealed that vestibular efferent neurons are located adjacent to, but are not included among, flocculus-projecting supragenual neurons. PRV transport from the vestibular labyrinth and cervical muscles also resulted in CNS infections. Virus propagation in situ provides specific connectivity information based on the functional transport across synapses. The findings support and extend anatomical data regarding vestibulo-olivo-cerebellar pathways.

Cytochrome oxidase histochemistry in Scarpa's ganglion after hemilabyrinthectomy.

Cytochrome oxidase histochemistry was studied in neurons in the vestibular ganglion in gerbils two weeks after hemilabyrinthectomy. This study measured the staining density in ganglion cells on both the lesioned and non-lesioned side of the brainstem. Cytochrome oxidase staining was significantly reduced in ganglion cells ipsilateral to the lesion. This decrease may have been related to the concomitant loss of spontaneous discharge and reduced energy demand for oxidative metabolism.

Translabyrinth electrical stimulation for the induction of immediate-early genes in the gerbil brainstem.

Brainstem immediate-early gene (IEG) protein expression was induced following applications of current to the labyrinth in unanesthetized gerbils. Electrode placement, stimulus polarity, current intensity and waveform, and anesthetics all significantly affect IEG expression patterns. Direct currents of different polarity applied across the labyrinth produced IEG expression in vestibular nuclei and inferior olivary neurons in patterns similar to those seen after hemilabyrinthectomy or hypergravity stimulation.

Encoding of head acceleration in vestibular neurons. I. Spatiotemporal response properties to linear acceleration.

1. Extracellular recordings were made in and around the medial vestibular nuclei in decerebrated rats. Neurons were functionally identified according to their semicircular canal input on the basis of their responses to angular head rotations around the yaw, pitch, and roll head axes. Those cells responding to angular acceleration were classified as either horizontal semicircular canal-related (HC) or vertical semicircular canal-related (VC) neurons. The HC neurons were further characterized as either type I or type II, depending on the direction of rotation producing excitation. Cells that lacked a response to angular head acceleration, but exhibited sensitivity to a change in head position, were classified as purely otolith organ-related (OTO) neurons. All vestibular neurons were then tested for their response to sinusoidal linear translation in the horizontal head plane. 2. Convergence of macular and canal inputs onto central vestibular nuclei neurons occurred in 73% of the type I HC, 79% of the type II HC, and 86% of the VC neurons. Out of the 223 neurons identified as receiving macular input, 94 neurons were further studied, and their spatiotemporal response properties to sinusoidal stimulation with pure linear acceleration were quantified. Data were obtained from 33 type I HC, 22 type II HC, 22 VC, and 17 OTO neurons. 3. For each neuron the angle of the translational stimulus vector was varied by 15, 30, or 45 degrees increments in the horizontal head plane. In all tested neurons, a direction of maximum sensitivity was identified. An interesting difference among neurons was their response to translation along the direction perpendicular to that that produced the maximum response ("null" direction). For the majority of neurons tested, it was possible to evoke a nonzero response during stimulation along the null direction always had response phases that varied as a function of stimulus direction. 4. These spatiotemporal response properties were quantified in two independent ways. First, the data were evaluated on the basis of the traditional one-dimensional principle governed by the "cosine gain rule" and constant response phase at different stimulus orientations. Second, the response gain and phase values that were empirically determined for each orientation of the applied linear stimulus vector were fitted on the basis of a newly developed formalism that treats neuronal responses as exhibiting two-dimensional spatial sensitivity. Thus two response vectors were determined for each neuron on the basis of its response gain and phase at different stimulus directions in the horizontal head plane.(ABSTRACT TRUNCATED AT 400 WORDS)

Two-dimensional spatiotemporal coding of linear acceleration in vestibular nuclei neurons.

Response properties of vertical (VC) and horizontal (HC) canal/otolith-convergent vestibular nuclei neurons were studied in decerebrate rats during stimulation with sinusoidal linear accelerations (0.2-1.4 Hz) along different directions in the head horizontal plane. A novel characteristic of the majority of tested neurons was the nonzero response often elicited during stimulation along the "null" direction (i.e., the direction perpendicular to the maximum sensitivity vector, Smax). The tuning ratio (Smin gain/Smax gain), a measure of the two-dimensional spatial sensitivity, depended on stimulus frequency. For most vestibular nuclei neurons, the tuning ratio was small at the lowest stimulus frequencies and progressively increased with frequency. Specifically, HC neurons were characterized by a flat Smax gain and an approximately 10-fold increase of Smin gain per frequency decade. Thus, these neurons encode linear acceleration when stimulated along their maximum sensitivity direction, and the rate of change of linear acceleration (jerk) when stimulated along their minimum sensitivity direction. While the Smax vectors were distributed throughout the horizontal plane, the Smin vectors were concentrated mainly ipsilaterally with respect to head acceleration and clustered around the naso-occipital head axis. The properties of VC neurons were distinctly different from those of HC cells. The majority of VC cells showed decreasing Smax gains and small, relatively flat, Smin gains as a function of frequency. The Smax vectors were distributed ipsilaterally relative to the induced (apparent) head tilt. In type I anterior or posterior VC neurons, Smax vectors were clustered around the projection of the respective ipsilateral canal plane onto the horizontal head plane. These distinct spatial and temporal properties of HC and VC neurons during linear acceleration are compatible with the spatiotemporal organization of the horizontal and the vertical/torsional ocular responses, respectively, elicited in the rat during linear translation in the horizontal head plane. In addition, the data suggest a spatially and temporally specific and selective otolith/canal convergence. We propose that the central otolith system is organized in canal coordinates such that there is a close alignment between the plane of angular acceleration (canal) sensitivity and the plane of linear acceleration (otolith) sensitivity in otolith/canal-convergent vestibular nuclei neurons.

Contribution of irregular semicircular canal afferents to the horizontal vestibuloocular response during constant velocity rotation.

1. The effects of constant anodal currents (100 microA) delivered bilaterally to both labyrinths on the horizontal vestibuloocular response (VOR) were studied in squirrel monkeys during steps of angular velocity in the dark. We report that bilateral anodal currents decreased eye velocity approximately 30-50% during the period of galvanic stimulation without a change in the time constant of VOR. The decrease in eye velocity, present during steps of angular velocity, was not observed during sinusoidal head rotation at 0.2, 0.5, and 1 Hz. The results suggest that responses from irregular vestibular afferents influence VOR amplitude during constant velocity rotation.

Role of irregular otolith afferents in the steady-state nystagmus during off-vertical axis rotation.

1. During constant velocity off-vertical axis rotations (OVAR) in the dark a compensatory ocular nystagmus is present throughout rotation despite the lack of a maintained signal from the semicircular canals. Lesion experiments and canal plugging have attributed the steady-state ocular nystagmus during OVAR to inputs from the otolith organs and have demonstrated that it depends on an intact velocity storage mechanism. 2. To test whether irregularly discharging otolith afferents play a crucial role in the generation of the steady-state eye nystagmus during OVAR, we have used anodal (inhibitory) currents bilaterally to selectively and reversibly block irregular vestibular afferent discharge. During delivery of DC anodal currents (100 microA) bilaterally to both ears, the slow phase eye velocity of the steady-state nystagmus during OVAR was reduced or completely abolished. The disruption of the steady-state nystagmus was transient and lasted only during the period of galvanic stimulation. 3. To distinguish a possible effect of ablation of the background discharge rates of irregular vestibular afferents on the velocity storage mechanism from specific contributions of the dynamic responses from irregular otolith afferents to the circuit responsible for the generation of the steady-state nystagmus, bilateral DC anodal galvanic stimulation was applied during optokinetic nystagmus (OKN) and optokinetic afternystagmus (OKAN). No change in OKN and OKAN was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in monkey horizontal semicircular canal afferent responses after spaceflight.

Extracellular responses from single horizontal semicircular canal afferents in two rhesus monkeys were studied after recovery from a 14-day biosatellite (COSMOS 2044) orbital spaceflight. On the 1st postflight day, the mean gain for 9 different horizontal canal afferents, tested using one or several different passive yaw rotation waveforms, was nearly twice that for 20 horizontal canal afferents similarly tested during preflight and postflight control studies. Adaptation of the afferent response to passive yaw rotation on the 1st postflight day was also greater. These results suggest that at least one component of the vestibular end organ (the semicircular canals) is transiently modified after exposure to 14 days of microgravity. It is unclear whether the changes are secondary to other effects of microgravity, such as calcium loss, or an adaptive response. If the response is adaptive, then this report is the first evidence that the response of the vestibular end organ may be modified (presumably by the central nervous system via efferent connections) after prolonged unusual vestibular stimulation. If this is the case, the sites of plasticity of vestibular responses may not be exclusively within central nervous system vestibular structures, as previously believed.