Potential pathways for intercellular communication within the calbindin subnucleus of the hamster suprachiasmatic nucleus.

In mammals, the suprachiasmatic nucleus (SCN) is the master circadian pacemaker. Within the caudal hamster SCN, a cluster of neurons containing the calcium binding protein, calbindin-D28K (CB), has been implicated in circadian locomotion. However, calbindin-immunoreactive (CB+) neurons in the calbindin subnucleus (CBsn) do not display a circadian rhythm in spontaneous firing [Eur J Neurosci 16 (2002) 2469]. Previously, we proposed that intercellular communication might be essential in integrating outputs from rhythmic (CB-) neurons and nonrhythmic (CB+) neurons to produce a circadian output in the intact animal. The primary aim of this study is to provide a neuroanatomical framework to better understand intercellular communication within the CBsn. Using reconstructions of previously recorded neurons, we demonstrate that CB+ neurons have significantly more dendrites than CB- neurons. In addition, CBsn neurons have dorsally oriented dendritic arbors. Using double-label confocal microscopy, we show that GABA colocalizes with CB+ neurons and GABA(A) receptor subunits make intimate contacts with neurons in the CBsn. Transforming growth factor alpha (TGFalpha), a substance shown to inhibit locomotion [Science 294 (2001) 2511], is present within the CBsn. In addition, neurons in this region express the epidermal growth factor receptor, the only receptor for TGFalpha. Lastly, we show that CB+ neurons are coupled to CB+ and CB- neurons by gap junctions. The current study provides a structural framework for synaptic communication, electrical coupling, and signaling via a growth factor within the CBsn of the hamster SCN. Our results reveal connections that have the potential for integrating cellular communication within a subregion of the SCN that is critically involved in circadian locomotion.

Modeling temporal behavior of postnatal cat retinal ganglion cells.

During development, mammalian retinal ganglion cells (RGCs) go through marked ontogenetic changes with respect to their excitable membrane properties. Voltage-clamp studies conducted in our laboratory have shown that the amplitude, voltage-dependence and kinetics of activation and inactivation (where present) of Na(+), K(+) and Ca(2+) conductances all exhibit developmental changes during a time when the firing patterns of mammalian ganglion cells shift from being transient to being predominantly sustained in nature. In order to better understand the contribution of each conductance to the generation of spikes and spiking patterns, we have developed a model based on our experimental data. For simplicity, we have initially used experimental data obtained from postnatal ganglion cells. At this age the ontogenetic changes observed in the characteristics of the various ionic currents are complete. Utilizing the methods adopted by Hodgkin and Huxley for the giant squid axon, we have determined rate equations for the activation and inactivation properties of the I(A), I(K dr), I(Na), I(Ca L), I(Ca N), and I(leak) currents in postnatal cat RGCs. Combining these with a simplified model of the calcium-activated potassium current (I(KCa)), we have solved and analysed the resulting differential equations. While spikes and spiking patterns resembling experimental data could be obtained from a model in which [Ca(2+)i] was averaged across the whole cell, more accurate simulations were obtained when the diffusion of intracellular Ca(2+) was modeled spatially. The resulting spatial calcium gradients were more effective in gating I(KCa), and our simulations more accurately matched the recorded amplitude and shape of individual spikes as well as the frequency of maintained discharges observed in mammalian postnatal RGCs.

Time-dependent changes in input resistance of rat hypoglossal motoneurons associated with whole-cell recording.

The effect of cellular dialysis associated with whole-cell recording was studied in 24 developing hypoglossal motoneurons in a rat brainstem slice preparation. In all cases, establishing whole-cell continuity with the electrode solution resulted in an increase in the input resistance measured in current clamp. The mean magnitude of this increase was 39.7% and the time course of the maximum effect was quite variable. There was no correlation found between the time to maximum effect and the magnitude of the increase in resistance. These data indicate that the passive membrane properties are not constant during whole-cell recording in mammalian motoneurons.

Development of intrinsic membrane properties in mammalian retinal ganglion cells.

It is now well established that retinal ganglion activity is essential to the normal development of the mammalian visual system. Moreover, it has been shown that the critical periods of activity occur well before the time when the retina is capable of detecting light. To better understand these activity-mediated events, patch-clamp studies have begun to examine the development of intrinsic membrane properties in isolated and intact retinal ganglion cells. Here we review the major findings of these studies and highlight the similarities in the functional development of ganglion cells in a number of mammalian species.

Activation and inactivation properties of voltage-gated calcium currents in developing cat retinal ganglion cells.

The correlated activity of developing retinal ganglion cells is essential for the reorganization and refinement of retinogeniculate projections. Previous studies have uncovered marked changes in the spiking properties of retinal ganglion cells during this period of reorganization; however, a full understanding of the changes in the underlying ionic conductances has yet to be obtained. To this end, the whole-cell configuration of the patch-clamp technique was used to record currents conducted by voltage-gated calcium channels in 83 dissociated cat retinal ganglion cells obtained from animals aged between embryonic day 34 and postnatal day 105. Calcium currents, magnified by using barium as the major charge carrier, were isolated by substituting choline for Na+ in the bathing solution and Cs+ for K+ in the electrode solution. Three voltage-gated Ca2+ conductances were identified based on their voltage dependence and kinetics of activation and inactivation: a transient low-voltage-activated conductance, a transient high-voltage-activated conductance and a sustained high-voltage-activated conductance. During the developmental period examined there were significant increases in the densities of all three conductances, as well as significant changes in some of their activation and inactivation properties. These findings, together with those reported previously for the voltage-gated Na+ and K+ conductances, are related to the generation of excitability in developing retinal ganglion cells during a period critical to the normal development of the visual system. Furthermore, while the sustained high-voltage-activated conductance was present in all of the retinal ganglion cells observed, only about 72% expressed the transient high-voltage-activated current. During the developmental period examined, there was also an increase in the proportion of cells expressing the transient low-voltage-activated conductance. This, along with our previous finding that retinal ganglion cells heterogeneously express different types of voltage-gated K+ channels, strongly suggests that the spiking patterns observed in different classes of retinal ganglion cell may be due, in part, to their intrinsic membrane properties.

Calcium-activated potassium conductances in retinal ganglion cells of the ferret.

Patch-clamp recordings were made from isolated and intact retinal ganglion cells (RGCs) of the ferret to examine the calcium-activated potassium channels expressed by these neurons and to determine their functional role in the generation of spikes and spiking patterns. Single-channel recordings from isolated neurons revealed the presence of two calcium-sensitive potassium channels that had conductances of 118 and 22 pS. The properties of these two channels were shown to be similar to those ascribed to the large-conductance calcium-activated potassium channel (BKCa) and small-conductance calcium-activated potassium channel (SKCa) channels in other neurons. Whole cell recordings from isolated RGCs showed that apamin and charybdotoxin (CTX), specific blockers of the SKCa and BKCa channels, respectively, resulted in a shortening of the time to threshold and a reduction in the hyperpolarization after the spike. Addition of these blockers also resulted in a significant increase in spike frequency over a wide range of maintained depolarizations. Similar effects of apamin and CTX were observed during current-clamp recordings from intact alpha and beta ganglion cells, morphologically identified after Lucifer yellow filling. About 20% of these neurons did not exhibit a sensitivity to either blocker, suggesting the presence of functionally distinct subgroups of alpha and beta RGCs on the basis of their intrinsic membrane properties. The expression of these calcium-activated potassium channels in the majority of alpha and beta cells provides a means by which the activity of these output neurons could be modulated by retinal neurochemicals.

The intrinsic temporal properties of alpha and beta retinal ganglion cells are equivalent.

BACKGROUND: Mammalian retinal ganglion cells have been traditionally classified on the basis of morphological and functional criteria, but as yet little is known about the intrinsic membrane properties of these neurons. This study has investigated these properties by making patch-clamp recordings from morphologically identified ganglion cells in the intact retina. RESULTS: The whole-cell configuration of the patch-clamp technique was used to assess the temporal tuning characteristics of alpha and beta cells, the two most extensively studied ganglion cell classes. Fourier analysis was used to examine discharge patterns in response to sinusoidal currents of different frequencies (1-50 Hz). With few exceptions, neurons responded in a stereotypic fashion to changes in temporal modulation, with their output initially increasing and then decreasing as a function of stimulus frequency. Moreover, peak responses in both cell classes were obtained at equivalent temporal frequencies. At high stimulus rates, response probability decreased, but the spikes remained phase-locked to the stimulus cycle, thereby enabling populations of cells to convey temporal information. A small number of ganglion cells did not show an appreciable decrease in output as a function of stimulus frequency, but these cells were not confined to either ganglion cell class. CONCLUSIONS: These findings provide the first evidence that the intrinsic temporal properties of alpha and beta cells are alike. Furthermore, the responses obtained to direct current injections were strikingly similar to those described previously with temporally modulated visual stimuli, suggesting that intrinsic membrane properties may shape the visual responses of alpha and beta cells to a larger degree than has been commonly assumed.

Concept of the notional person in the assessment of hearing disability.

Medicolegal assessment in relation to a claim for hearing damage requires an equitable baseline against which to compare the state of hearing of the claimant. This baseline, or 'notional person', should match the claimant in every respect save for the cause of the hearing loss. The amount of compensation is determined from the difference between the percentage disability of the claimant and that of the 'notional person'. These percentages are, in turn, calculated from the respective hearing threshold levels. In the particular case of occupational noise-induced hearing loss there are, in principle, two ways of proceeding. One way is to estimate directly the noise-induced threshold shift which would result from the noise exposure and to subtract this from the claimant's measured threshold level; in practice the noise exposure is rarely known with the necessary accuracy. The alternative method is to construct the notional person's threshold level by adding together all the non-compensable components of hearing loss known to exist in the claimant. These may include: conductive hearing loss together with any sensorineural sequelae, sensorineural loss specifically attributable to identifiable accident or disease, pure biological ageing, any pathological overlay due to deficiencies of aural or general health, the effect of non-occupational noise, and the socioeconomic status of the claimant. Once these components are summed, whatever remains unaccounted for is deemed to be due to the occupational noise. An International Standard (ISO 7029) exists for the biological ageing component but it is specific to 'otologically normal' persons and on its own does not account for all the time-dependent hearing losses found in a typical claimant. Moreover, the term 'otologically normal', although defined in the relevant Standards, leads to varying interpretations, even to misunderstandings. In practice, the equitable baseline for the disability assessment is most often a 'typical' rather than an 'otologically normal' person in the Standards sense. This paper offers guidance for estimating the combined time-dependent threshold shift in these commonly occurring cases, based on the use of published survey data. It is recommended that a presumption of 'typical hearing' should be made unless the claimant in question can be specifically verified as 'otologically normal'.

Properties of K+ conductances in cat retinal ganglion cells during the period of activity-mediated refinements in retinofugal pathways.

During ontogeny retinal ganglion cells manifest pronounced changes in excitable membrane properties. To further our understanding of the ionic conductances underlying such functional changes, the whole-cell voltage-clamp variation of the patch-clamp technique was used to record potassium currents in 220 ganglion cells dissociated from cat retinas ranging in age from embryonic day 31 to postnatal day 10. Potassium currents were isolated by blocking voltage-gated Na+ and Ca2+ currents with tetrodoxin (TTX) and CoCl2 respectively and were characterized by their pharmacology, kinetics and voltage-dependence of activation and inactivation. In all cases, a combination of three currents accounted for the total outward calcium-independent K+ current: (i) a steady linear conductance; (ii) a voltage-gated transient current, IA, and (iii) a voltage-gated sustained current, IK. Both voltage-gated currents were affected by the application of 4-aminopyridine and tetraethylammonia (TEA): IA showed a greater sensitivity to 4-aminopyridine, while IK was more sensitive to TEA. Both voltage-gated currents were present throughout the developmental period examined; however, the percentage of retinal ganglion cells (RGCs) expressing IA showed a marked decline from 82% at E31 to 45% at postnatal ages. During this developmental period there was an increase in the density of the two voltage-gated and the linear conductance. Additionally, with maturation, significantly slower inactivation kinetics were observed for IK. These findings, and our previous results dealing with maturational changes in the TTX-sensitive voltage-gated Na current, are related to the generation of excitability in developing retinal ganglion cells. Furthermore, the presence of cells with and without transient K+ conductance throughout development suggests that the different spiking patterns observed in RGC classes may be partially due to differences in their membrane properties.

Free jejunal interposition reconstruction after pharyngolaryngectomy: 201 consecutive cases.

BACKGROUND: Reconstruction of tubular defects following pharyngolaryngectomy has required complicated surgery with high perioperative morbidity and mortality. Free jejunal interposition provides an excellent reconstruction with potential for lower immediate complications and better long-term results than other procedures. METHODS: A total of 201 consecutive free jejunal interpositions were performed following pharyngolaryngectomy between 1977 and 1993. Operative details, complications, and outcome were prospectively documented. RESULTS: Perioperative mortality was low (4.5%) and microvascular success rate high (97%), although a small number of late failures were recorded. Average time until swallowing postoperatively was 11 days, and 92% of patients could maintain full nutrition. Voice rehabilitation was mentioned, and increasingly good results are being obtained. Complication rates for the neck (17%) and the abdomen (2.5%) were also low. There were no problems with excess mucus production or reflux. Radiation effect on the jejunal conduit was not detrimental to long-term patency of the vascular anastomoses or to function as a conduit. CONCLUSIONS: Comparison with other published techniques permits the contention that a free jejunal interposition is the reconstruction of choice after pharyngolaryngectomy.

Imaging the ovine heparin-protamine interaction with 111In-protamine.

Protamine reversal of heparin anticoagulation occasionally induces the release of thromboxane into plasma with catastrophic pulmonary hypertension. To examine the site of neutralization, we labeled protamine sulfate with 111In and compared activity scans after administration of labeled protamine in unheparinized and heparin-anticoagulated sheep. Protamine administration in sheep without prior heparinization did not cause thromboxane release, pulmonary hypertension, or significant leukopenia, and 111In-protamine was rapidly cleared from the lungs (half time 0.48 +/- 0.08 min). Neutralization of heparin anticoagulation by labeled protamine produced elevated plasma thromboxane, pulmonary vasoconstriction, leukopenia, and prolonged pulmonary clearance of 111In-protamine (half time 3.32 +/- 0.43 min). In rats, protamine reversal of heparin anticoagulation did not induce either thromboxane synthesis or pulmonary hypertension, and 111In-protamine cleared rapidly from the lungs. Thus the ovine heparin-protamine reaction produces concomitant pulmonary sequestration of heparin-protamine complexes, thromboxane release, and pulmonary vasoconstriction; this did not occur in the rat. The lung specificity of the reaction and interspecies differences suggest that ovine pulmonary intravascular macrophages may be activated by heparin-protamine complexes to release thromboxane and provoke acute pulmonary vasoconstriction.

Temperature dependence of the light response in rat rods.

1. The effects of temperature on the light responses of rat rods have been investigated over the range 17-40 degrees C. 2. The amplitude of the light-sensitive current increased with temperature with a mean temperature coefficient (Q10) of 2.47. 3. The amplitude of the Na(+)-Ca2+, K+ exchange current decreased with temperature when expressed as a fraction of the light-sensitive current, showing that the light-sensitive channel becomes less permeable to calcium as the temperature is raised. The time constant of relaxation of the exchange current was little affected by temperature. 4. The flash intensity required to give a half-saturating response increased with temperature with a mean Q10 of 1.68. 5. The responses to single photoisomerizations were determined from amplitude histograms of the responses to dim-flash trains. The amplitude of the response to a single photoisomerization decreased with temperature when expressed as a fraction of the light-sensitive current, but the change was not sufficient to account for the overall decrease in sensitivity. 6. The fraction of dim flashes that produced a photoisomerization decreased with temperature. This decrease in photon capture efficiency together with the decrease in the relative size of the single photon event fully accounts for the observed change in sensitivity. 7. The speed of the falling phase of the dim-flash response was accelerated more by warming than the rising phase, and it was therefore not possible to superimpose light responses at different temperatures by a simple change in time scale.

Quantification of hearing disability for medicolegal purposes based on self-rating.

Current practice for medicolegal assessment of individuals entails the use of an impairment measure obtained from average hearing threshold levels as a surrogate for hearing disability, and conversion of the surrogate to disability via a formula. Several different formulae are in use, but none is based explicitly on experimental data. To address this lack of empirical foundation in the assessment process, numerical self-ratings of hearing ability by 2058 subjects with a wide range of hearing threshold levels who had taken part in the National Study of Hearing in the UK were analysed to examine their relation to average hearing threshold level. The relation between self-rated hearing disability (the complement of self-rated hearing ability) and hearing threshold level was found to be sigmoid in form, and could be closely modelled by a modified Gompertz function. Functions for the median, upper quartile and lower quartile disability ratings with hearing threshold level are presented in graphical, parametric and tabular form. The median function gives a quantitative foundation for medicolegal assessments.

Background noise in rooms used for pure-tone audiometry in disability assessment.

Background noise limits for audiometry are determined by the effects of masking and are specified in international standards. The standards provide for audiometric testing over a range of audiometric frequencies extending down to 500 Hz or lower. The lowest frequency of testing is an important factor determining the admissible noise, and for certain applications it is appropriate to consider the limits applicable to testing over a more restricted range. Assessment of hearing disability in the UK is generally based on a consideration of pure-tone hearing threshold levels in the frequency range 1 kHz upwards. A modification of the standardized noise limits is proposed which allows some relaxation appropriate to this higher minimum frequency. For air-conduction audiometry, these modifications affect only the permissible background noise in the frequency range below 1 kHz. Where bone-conduction audiometry is required in order to quantify a conductive component of the hearing loss, the measurements need to be made on both ears with the non-test ear masked in both cases; the external background noise will thus only be heard monaurally and this justifies a correction to the noise limits compared with those appropriate to bone-conduction audiometry without masking.

Long-term repeatability of the pure-tone hearing threshold and its relation to noise exposure.

Audiometric repeatability is examined for a population of 356 male and female industrial employees (712 ears), sub-divided according to the amount of previous noise exposure. Re-tests were carried out after an interval averaging 13 months. After a similar interval, 150 of the subjects gave a third audiogram. Fixed-frequency self-recording audiometry was employed, using common equipment and procedures throughout. Results are presented as distributions of the algebraic test-re-test differences for each frequency and for the frequency combination 1-2-3 kHz. Repeatability was best at 1 and 2 kHz and substantially poorer at 6 kHz. Cumulative distributions of the signless differences show that more than 50% of initial hearing threshold levels repeated to better than 5 dB. For all frequencies except 6 kHz the mean values of signless test-re-test differences for the various sub-groups were between 3.4 and 5.6 dB; at 6 kHz the values were between 5.8 and 7.8 dB. In the case of the three-frequency average, the mean differences were reduced to between 2.8 and 3.8 dB. A few repeats (about one in 200) stand out as clearly anomalous; these occur only in isolation and mainly at the higher frequencies. The performance of groups classified by the amount of their previous noise exposure did not differ significantly. In the case of those tested three times, the repeatability over the second inter-test interval showed at most a marginal improvement compared with the first interval.

The limited accuracy of bone-conduction audiometry: its significance in medicolegal assessments.

Accurate bone-conduction testing with masking is always difficult, but for clinical purposes limited accuracy suffices. However, when assessing claimants for compensation, extreme care is needed since even small apparent air-bone gaps are sometimes translated into financial abatement. This paper sets out the stringent test conditions required to achieve adequate precision. It also indicates the inaccuracies inherent in such tests, and recommends procedures for interpreting the significance of bone-conduction thresholds.

Development of the light response in neonatal mammalian rods.

The sensitivity to light is low in many neonatal mammals when compared with that in the adult. In human infants at one month of age, for example, the dark-adapted sensitivity for detection of large stimuli is 50 times lower than in the adult, and in rats the overall sensitivity of the neonatal retina is also low compared with the adult. This low sensitivity in the neonate has been attributed to a number of factors, but the possibility that the photoreceptors themselves might be an important limitation on the overall visual sensitivity has not so far been clearly established. Here we record the light response of single neonatal rat rods and find that the sensitivity is considerably lower than in the adult. The response to a single photoisomerization is normal in the neonate, and the sensitivity deficit can therefore be attributed to a low level of functional rhodopsin. Opsin, the protein component of rhodopsin, must be present in normal amounts, as the sensitivity can be restored to adult levels by treating the retina with 9-cis retinal, an active homologue of the native chromophore 11-cis retinal. The low sensitivity of photoreceptors in the neonate can therefore be attributed mainly to a low concentration of 11-cis retinal in the developing retina.