Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.¹³. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia^1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain⁴. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving^5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats^7-9 to mouse^10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.     

High-precision coding in visual cortex

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Neurons discovered in male Helicoverpa zea antennae that correlate with pheromone-mediated attraction and interspecific antagonism

Responses of single receptor neurons in the antennae of male Helicoverpa zea to sex pheromone components and to behavioral antagonists were recorded using a cut-sensillum extracellular recording technique. Three types of sensilla were identified from sampling 325 male-specific sensilla trichodea located at the lateral edge of antennomeres. The majority of these sensilla (71%) contained a receptor neuron tuned to the principal sex pheromone component (Z)-11-hexadecenal. A second sensillar type (10%) contained a receptor neuron that responded only to (Z)-9-tetradecenal. A third sensillar type (19%) contained a large-spiking neuron tuned to the secondary pheromone component (Z)-9-hexadecenal, but this neuron also could be stimulated to equivalent spike frequencies by the same emitted amounts of (Z)-9-tetradecenal. A smaller-spiking neuron in this sensillar type responded to two compounds known to act only as behavioral antagonists, (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate, and to (Z)-9-tetradecenal. Cross-adaptation studies confirmed the presence of one large- and one small-spiking neuron in the third sensillar type. Dose-response studies correlated to collected stimuli amounts showed that the large-spiking neuron in the third sensillar type was equally tuned to (Z)-9-hexadecenal and (Z)-9-tetradecenal, whereas the smaller-spiking neuron was far more sensitive to (Z)-11-hexadecen-1-ol and to (Z)-11-hexadecenyl acetate than to (Z)-9-tetradecenal. Accepted: 29 September 1997     

Neurophysiologic measurement of continuity in the sleep of fetuses during the last week of pregnancy and in newborns

Our aim was to measure the correlation between fetal electrocardiographic (FECG) recordings of low-risk pregnancies and polysomnographic (PSG) study parameters in low-risk infants born at term as a measurement of perinatal sleep-development continuity.We designed a short, prospective, observational follow-up of physiologic parameters between fetuses and newborns. We studied 10 fetuses from low-risk pregnant female out-patients and the same subjects as low-risk newborns delivered at term. Fetal state (FS) was defined in FECG recordings reassembling the following: fetal state I (quiet sleep or QS); fetal state II (active sleep or AS); fetal state III (quiet waking), and fetal state IV (active waking). Percentages of AS, QS, and wakefulness in PSG studies of newborns were also determined.Comparisons of FS I with QS showed a significant reduction in QS, while comparison of FS II with AS showed significant reduction in AS. Negative correlations were found between FS I with QS, and FS II with AS. Number of cycles in FECG recordings and PSG sleep cycles also demonstrated significant correlation.In conclusion our data showed partial but significant sleep function continuity from fetal to neonatal period.     

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.     

A Spatial Map of Onset and Sustained Responses to Speech in the Human Superior Temporal Gyrus

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Inhomogeneous Encoding of the Visual Field in the Mouse Retina

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Electrophysiological and behavioural evidence for a fourth sex pheromone component in the turnip moth, Agrotis segetum

Abstract. In addition to the pheromone components (Z)-5-decenyl, (Z)-7-dodecenyl and (Z)-9-tetradecenyl acetate (Z5-10:OAc, Z7-12:OAc and Z9-14:OAc), it has previously been shown that the sex pheromone gland of the turnip moth, Agrotis segetum (Lepidoptera: Noctuidae Schiff) contains 10:OAc, 12:OAc, Z5-12:OAc, Z9-12:OAc, 11–12:OAc, Z5-14:OAc, Z7-14:OAc and Z11-16:OAc. To find out whether any of these additional compounds is involved in the sex pheromone communication in A. segetum, a comprehensive electro-physiological and behavioural investigation was conducted. Single-sensillum recordings on male antennae revealed three subtypes of sensilla among the previously so-called Z5-10:OAc sensilla. One subtype was identified having one receptor neurone (A) that responded to Z5-10:OAc with a large spike amplitude and another neurone (B) that responded to (Z)-5-decenol (Z5-10:OH) with a small spike amplitude. In another subtype the B neurone responded to Z5-12:OAc and sometimes also to 27-12:OAc and 10:OAc, in addition to responding to Z5-10:OH. In a third subtype the A neurone responded to all acetates identified from the female pheromone gland, whereas the small spike amplitude neurone was tuned to Z5-10:OH. A flight tunnel assay showed that blends composed of nine, eight or seven compounds were equivalent to the previously identified three-component pheromone blend in eliciting male behavioural responses. In field trapping tests, blends of eleven, nine or seven compounds did, however, catch significantly more moths than the three-component blend. Further assays showed that only 25- 12:OAc could significantly increase the catch numbers when added to the three-component blend, and thus qualified as a fourth pheromone component in A. segerum. The behavioural significance of additional female-produced acetates — for which males possess antennal receptors — is suggested, but may be impossible to confirm because of ‘diminishing returns’ when trying to refine a multicomponent pheromone further.     

Channel gating in the absence of agonist by a homooligomeric molluscan GABA receptor expressed inXenopus oocytes from a cloned cDNA

We have previously described the isolation of a complementary DNA (cDNA) from the freshwater molluscLymnaea stagnalis encoding a polypeptide that exhibits 50% identity to the ß-subunits of vertebrate -aminobutyric acid (GABA) type A (GABA_A) receptor. When expressed inXenopus laevis oocytes fromin vitro-transcribed RNA, the snail subunit forms functional homo-oligomeric receptors possessing chloride-selective ion channels. In recordings from voltage-clamped oocytes held at –60 mV, GABA induced an inward current, whereas application of the chloride-channel blocker picrotoxin (in the absence of agonist) elicited an apparent outward current. Single channel recordings obtained from cell-attached patches have revealed a single population of 20 pS channels, with an open probability greater than 90% (at a pipette potential of –100 mV) in the absence of GABA. The relationship between single channel current and pipette potential was linear over the studied range (–100 mV to +60 mV), but the open probability was less for hyperpolarizations than for depolarizations. The spontaneous channel openings were blocked by micromolar concentrations of picrotoxin. Functional hetero-oligomeric receptors were formed when the molluscan subunit was co-expressed in oocytes with the bovine GABA_A receptor 1-subunit, but the channels gated by these receptors did not open spontaneously.     

Single cell recordings reveal hydroxydanaidal as the volatile compound attracting insects to pyrrolizidine alkaloids

Employment of electrophysiology in combination with simple chemical techniques elucidated the volatile which permit the localization of sources of pyrrolizidine alkaloids (PAs) by insects exploiting these secondary plant metabolites.Single cell recordings in Rhodogastria moths (Arctiidae) revealed a physiologically clearly separable type of antennal sensilla basiconica (SB II) which responds to headspace air of certain PAs (e.g. monocrotaline), but not to a great variety of odorants. However, stimulus sources of 1 g were required to elicit responses and the maximum frequency (obtained with stimulus sources of 5 mg) was only 50 imps/s. This suggested the occurrence of small amounts of airborne PA degradation products.Extraction and hydrolysis experiments in combination with thin-layer chromatography and using the sensory responses of antennal receptor cells as biological detectors eventually demonstrated that the dihydropyrrolizine hydroxydanaidal emanates in small amounts from sources of those PAs which contain retronecine and heliotridine, respectively, as necine moiety. This substance was also implicated as the volatile mediating attraction of the insects to PA-containing plants, as well as to artificial PA-baits. With respect to the high sensitivity and specificity of SB II-receptor cells to hydroxydanaidal, in particular to its R(-)-enantiomer, they are analogous to the well studied receptor cells for sex-attractant pheromones in Lepidoptera. Similar results were obtained with Danaus (Danainae) and Euchromia (Ctenuchiidae). Initial behavioural tests have proven the attractive power of hydroxydanaidal for PA-insects and thus corroborate our interpretation of the electrophysiological findings.

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Zusammenfassung Elektrophysiologische und einfache chemische Verfahren erlaubten die Eingrenzung und Identifikation des reizwirksamen Prinzips, welches Insekten, die Pyrrolizidin-Alkaloide (PA) nutzen, die Orientierung zu PA-Quellen ermöglicht.Einzelzellableitungen von antennalen Sensilla basiconica bei Rhodogastria (Lepidoptera: Arctiidae) zeigten einen morphologisch nicht unterscheidbaren physiologischen Typ, dessen Rezeptorzellen sich durch eine sehr geringe Spontanaktivität auszeichneten und ausschließlich auf PA-Duft reagierten (SB II). Allerdings wurden (z. B. mit Monocrotalin) Reizquellen-Beladungen von 1 g benötigt, um überschwellige Antworten auszulösen, und die maximal erreichte Aktivität (bei Reizung mit 5 mg) betrug lediglich 50 Imp./s; dies deutete auf das Auftreten von kleinen Mengen eines volatilen Abbauprodukts von PA hin.SB II wurden als biologische Detektoren verwendet, um — kombiniert mit Dünnschichtchromatographie — die Reizwirksamkeit von Produkten von Hydrolyse- und Extraktions-Experimenten mit PA zu testen. Auf diese Weise konnte schließlich gezeigt werden, daß von PA mit Retronecin bzw. Heliotridin als Necin geringe Mengen des Dihydropyrrolizins Hydroxydanaidal (Fig. 1G, H) ausgehen. Synthetisches Hydroxydanaidal stellte sich als der bestwirksame Reiz für SB II-Rezeptorzellen heraus, die bzgl. Spezifität und Sensitivität (insbesondere für R(-)-Hydroxydanaidal) den gut untersuchten Rezeptoren für weibliche Sexualpheromone bei Nachtfaltern vergleichbar sind. Entsprechende Ergebnisse wurden auch mit Danaus (Danainae) und Euchromia (Ctenuchiidae) gewonnen. Erste Verhaltensversuche beweisen die Lockwirkung von Hydroxydanaidal für PA-Insekten und bestätigen die Interpretation der elektrophysiologischen Befunde.