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Mammals are constantly exposed to exogenous and endogenous influences that affect their behaviour and daily activity. Light and temperature, as well as anthropogenic factors such as husbandry routines, visitors, and feeding schedules are potential influences on animals in zoological gardens. In order to investigate the effects of some of these factors on animal behaviour, observational studies based on the analyses of activity budgets can be used. In this study, the daily and nightly activity budgets of six lions (Panthera leo) and five cheetahs (Acinonyx jubatus) from four EAZA institutions were investigated. Focused on the influencing factor light and feeding, we analysed these activity budgets descriptively. Behaviour was recorded and analysed during the winter months over an observation period of 14 days and 14 nights using infrared-sensitive cameras. Our results show that lions and cheetahs exhibit activity peaks at crepuscular and feeding times, regardless of husbandry. Thus, lions in captivity shift nocturnal behaviour familiar from the wild to crepuscular and diurnal times. In cheetahs, in contrast, captive and wild individuals show similar 24 h behavioural rhythms. The resting behaviour of both species is more pronounced at night, with cheetahs having a shorter overall sleep duration than lions. This study describes the results of the examined animals and is not predictive. Nevertheless, the results of this study make an important contribution to gaining knowledge about possible factors influencing the behaviour of lions and cheetahs in zoos and offer implications that could be useful for improving husbandry and management.

In the past decades, zoos have increasingly developed into conservation and education centers and today make an important contribution to environmental education. In this context, this study invest...

Locating a vocalizing animal can be useful in many fields of bioacoustics and behavioral research, and is often done in the wild, covering large areas. In zoos, however, the application of this method becomes particularly difficult, because, on the one hand, the animals are in a relatively small area and, on the other hand, reverberant environments and background noise complicate the analysis. Nevertheless, by localizing and analyzing animal sounds, valuable information on physiological state, sex, subspecies, reproductive state, social status, and animal welfare can be gathered. Therefore, we developed a sound localization software that is able to estimate the position of a vocalizing animal precisely, making it possible to assign the vocalization to the corresponding individual, even under difficult conditions. In this study, the accuracy and reliability of the software is tested under various conditions. Different vocalizations were played back through a loudspeaker and recorded with several microphones to verify the accuracy. In addition, tests were carried out under real conditions using the example of the giant otter enclosure at Dortmund Zoo, Germany. The results show that the software can estimate the correct position of a sound source with a high accuracy (median of the deviation 0.234 m). Consequently, this software could make an important contribution to basic research via position determination and the associated differentiation of individuals, and could be relevant in a long-term application for monitoring animal welfare in zoos.

Upon a drastic decline of the giraffe population in the wild, conservation efforts and therefore the role of zoos have become more important than ever. With their unique opportunities, zoos provide excellent conditions to study animal behavior, expanding the knowledge about the giraffe's behavior repertoire and their ability to adapt. This study therefore examined the nocturnal behavior of 63 giraffe living in 13 different EAZA zoos across Germany and the Netherlands. Giraffe were observed and videos recorded via infrared sensitive cameras during the winter seasons 2015-2018. The observation period spanned nightly from 17:00 to 7:00. Thus, 198 nights, with a total of 2772 h were recorded and analyzed. Linear mixed models were then used to assess potential biological and environmental factors influencing behavior during the dark phase. Results show that individual variables such as age, subspecies and motherhood determined nocturnal activity and sleep behavior most. Among the variables studied, husbandry conditions and environmental factors complying with EAZA standards had no influence on the giraffe's nocturnal behavior. By combining nocturnal activity analyses and an assessment of potential influencing factors, our findings present a holistic approach to a better understanding of captive giraffe behavior and allow for management implications.

1. The description and analysis of animal behaviour over long periods of time is one of the most important challenges in ecology. However, most of these studies are limited due to the time and cost required by human observers. The collection of data via video recordings allows observation periods to be extended. However, their evaluation by human observers is very time-consuming. Progress in automated evaluation, using suitable deep learning methods, seems to be a forwardlooking approach to analyse even large amounts of video data in an adequate time frame. 2. In this study we present amulti-step convolutional neural network system for detecting animal behaviour states, which works with high accuracy. An important aspect of our approach is the introduction of model averaging and post-processing rules to make the system robust to outliers. 3. Our trained system achieves an in-domain classification accuracy of >0.92, which is improved to >0.96 by a postprocessing step. In addition, the whole system performs even well in an out-of-domain classification task with two unknown types, achieving an average accuracy of 0.93. We provide our system at https://github.com/Klimroth/Video-Action-Classifier-for-African-Ungulates-in-Zoos/tree/main/mrcnn_based so that interested users can train their own models to classify images and conduct behavioural studies of wildlife. 4. The use of a multi-step convolutional neural network for fast and accurate classification of wildlife behaviour facilitates the evaluation of large amounts of image data in ecological studies and reduces the effort of manual analysis of images to a high degree. Our system also shows that post-processing rules are a suitable way to make species-specific adjustments and substantially increase the accuracy of the description of single behavioural phases (number, duration). The results in the out-of-domain classification strongly suggest that our system is robust and achieves a high degree of accuracy even for new species, so that other settings (e.g. field studies) can be considered.

This research evaluates the development of three environmental knowledge dimensions of secondary school students after participation in a singular 1-day outdoor education programme. Applying a cross-national approach, system, action-related and effectiveness knowledge levels of students educated in Germany and Singapore were assessed before and after intervention participation. Correlations between single knowledge dimensions and behaviour changes due to the environmental education intervention were examined. The authors applied a pre-, post- and retention test design and developed a unique multiple-choice instrument. Results indicate significant baseline differences in the prevalence of the different knowledge dimensions between subgroups. Both intervention subsamples showed a low presence of all baseline knowledge dimensions. Action-related knowledge levels were higher than those of system and effectiveness knowledge. Subsample-specific differences in performed pro-environmental behaviour were also significant. Both experimental groups showed significant immediate and sustained knowledge increases in the three dimensions after programme participation. Neither of the two control cohorts showed any significant increase in any knowledge dimension. Effectiveness knowledge improved most. The amount of demonstrated environmental actions increased significantly in both intervention groups. Both control cohorts did not show shifts in environmental behaviour. Yet, only weak correlations between any knowledge dimension and behaviour could be found.

This study employs a uniquely multi-factorial, large-scale design to investigate baseline differences and the effects of a singular outdoor educational program on environmental attitudes, knowledge...

The present study assessed the diurnal variation in salivary cortisol in captive African elephants during routine management (baseline) and in relation to a potential stressor (translocation) to evaluate to what extent acute stress may affect diurnal cortisol patterns. Under baseline conditions, we collected morning and afternoon saliva samples of 10 animals (three zoos) on different days in two study periods (n = 3-10 per animal, daytime and period). Under stress conditions, we sampled the transported cow (newcomer) and the two cows of the destination zoo before and after the transport in the morning and afternoon (n = 3-9 per animal, daytime and transport phase), as well as after the first introduction of the newcomer to the bull (n = 1 per animal). Cortisol was measured in unextracted samples by enzyme immunoassay. Under baseline conditions, we observed the expected diurnal variation with higher cortisol levels in the morning than in the afternoon. Under stress conditions, neither a significant difference between pre- and posttransport, nor between morning and afternoon levels was found. The percentage difference between morning and afternoon cortisol after the transport, however, was remarkably lower than before the transport in the newcomer potentially indicating a stress response to familiarization. Saliva samples taken immediately after the introduction of the newcomer to the bull revealed a marked cortisol increase. Our findings indicate that stressors may disturb the diurnal cortisol rhythm. Furthermore, provided that samples can be collected promptly, salivary cortisol is a useful minimally invasive measure of physiological stress in the African elephant.

Nature connectedness counts as a crucial predictor of pro-environmental behavior. For counteracting today’s environmental issues a successful re-connection of individuals to nature is necessary. Besides the promotion of knowledge transfer the aim of the educational program presented in this study is to connect students to their environment. This research explores the impact of an outdoor environmental education program on primary and secondary school students’ nature connectedness with regard to the extent of their nature experience and participant age. The intervention was implemented in two durations: one-day and five-days. Participants were divided into four subsamples from seven up to 18 years of age. Findings suggest that both intervention types evoke immediate shifts towards a stronger nature connectedness among students (p

The interaction of internal clocks and environmental conditions determines the daily behavioural rhythm of an animal. Due to the strong influence of light, these circadian rhythms oscillate in mammals over a cycle length of about 24 h, equivalent to the daily light-dark cycle. The relation between activity and rest within this cycle is species-specific and age dependent. Since strong deviations from existing rhythms can harm health seriously, the observation of animal behaviour using activity budgets is a common tool to assess welfare. This study therefore investigated the nightly activity budget of 63 giraffe (Giraffa camelopardalis) from 13 EAZA institutions. The nightly behaviour was recorded and analysed in the winter seasons 2015–2018 over an observation period from 17:00 to 7:00 h for 10–14 nights, using infrared sensitive cameras. To analyse rest-activity rhythms of three age classes of giraffe during the dark phase of the night, linear mixed models were applied. Our results show that activity increases with increasing age of giraffe. Differences between age classes were further found for the time spent on rapid eye movement (REM) sleep, with juvenile and sub-adult giraffe spending more time in this specific state than adults. Analyses of behavioural rhythmicity indicated a rest-activity cycle length of about four hours, with age determining the rhythmic curve. Overall, this large-scale study confirms a strong age effect for both the nocturnal activity budget and the rest-activity rhythm in giraffe. The results of this study provide an important contribution to the continuous improvement of husbandry and management for zoo housed giraffe.

Summary Purpose: Despite the consistent observation that γ-aminobutyric acid A (GABAA) receptors mediate excitatory responses at perinatal stages, the role of the GABAergic system in the generation of neonatal epileptiform activity remains controversial. Therefore, we analyzed whether tonic and phasic GABAergic transmission had differential effects on neuronal excitability during early development. Methods: We performed whole cell patch-clamp and field potential recordings in the CA3 region of hippocampal slices from immature (postnatal day 4–7) rats to analyze the effect of specific antagonists and modulators of tonic and phasic GABAergic components on neuronal excitability. Key Findings: The GABAergic antagonists gabazine (3 μm) and picrotoxin (100 μm) induced epileptiform discharges, whereas activation of GABAA receptors attenuated epileptiform discharges. Under low-Mg2+ conditions, 100 nm gabazine and 1 μm picrotoxin were sufficient to provoke epileptiform activity in 63.2% (n = 19) and 53.8% (n = 26) of the slices, respectively. Whole-cell patch-clamp experiments revealed that these concentrations significantly reduced the amplitude of phasic GABAergic postsynaptic currents but had no effect on tonic currents. In contrast, 1-μm 4,5,6,7-tetrahydroisoxaz-olo[5,4-c]-pyridin-3-ol (THIP) induced a tonic current of −12 ± 2.5 pA (n = 6) and provoked epileptiform discharges in 57% (n = 21) of the slices. Significance: We conclude from these results that in the early postnatal rat hippocampus a constant phasic synaptic activity is required to control excitability and prevent epileptiform activity, whereas tonic GABAergic currents can mediate excitatory responses. Pharmacologic intervention at comparable human developmental stages should consider these ambivalent GABAergic actions.

SUMMARYThe hyperpolarisation-activated cation current (Ih) has been described in many vertebrate and invertebrate species and cell types. In neurons, Ih is involved in rhythmogenesis, membrane potential stabilisation and many other functions. In this work, we investigate the distribution and functional properties of Ih in identified leech neurons of intact segmental ganglia. We found Ih in the mechanosensory touch (T), pressure (P) and noxious (N) neurons, as well as in Retzius neurons. The current displayed its largest amplitude in P neurons and we investigated its biophysical and pharmacological properties in these cells. Ih was half-maximally activated at –65 mV and fully activated at –100 mV. The current mutually depended on both Na+ and K+ with a permeability ratio pNa/pK of ∼0.21. The reversal potential was approximately –35 mV. The time course of activation could be approximated by a single time constant of ∼370 ms at –60 mV, but required two time constants at –80 mV of ∼80 and ∼560 ms. The current was half-maximally blocked by 0.3 mmol l–1 Cs+ but was insensitive to the bradycardic agent ZD7288. The physiological function of this channel could be a subtle alteration of the firing behaviour of mechanosensory neurons as well as a stabilisation of the resting membrane potential.

International audience; A very small liquid‐membrane based pH probe (less than 500 nm diameter) has been implemented in scanning electrochemical microscopy (SECM) equipped with a nonoptical shear force‐based distance control. This constant‐distance mode pH microscopy was tested over a complex microcavity (50 μm diameter, 8 μm deep) possessing three individually‐addressable gold electrodes: a microdisk at the bottom, a microcrescent ring 4 μm away and along the wall, and a planar macroelectrode around the rim at the top. All the experiments were performed in a buffer of pH 6.22. The pH inside the microcavity was modified locally by applying negative potential (reduction of O2 and H2O to produce OH−) either at the ring or disk electrodes. The shear force‐sensitive signal permitted the precise determination of the tip‐to‐sample distance. Moreover, operating in constant‐distance mode, the tip‐to‐sample distance was kept constant (less than 200 nm) while scanning the electrode over the sample. The simultaneous collection of both topographic and potentiometric information allowed a better understanding of the complex electrochemical processes occurring within the microcavity. Since potentiometry is not disturbing diffusion profiles in front of active electrodes constant‐distance scanning potentiometry can be applied for the visualization of processes occurring in close proximity to electrode surfaces. This is especially important for the elucidation of properties of redox recycling systems often applied in amplified electrochemical assays.

The osmolarity of the extracellular space (ECS) compartment is an important factor determining the excitability of neuronal tissue. In the adult hippocampus an important role of osmolarity and ECS diffusion parameters on the susceptibility to epileptic events is well established, but the influence of hypo- and hyperosmolar conditions on the immature hippocampus remains elusive. To investigate the influence of osmolarity on epileptiform activity, extracellular field potentials were recorded in the CA3 region of hippocampal slices of immature (postnatal days 4-7) Wistar rats. The ECS diffusion parameters were determined by the real-time tetramethylammonium (TMA+) iontophoretic method with ion-selective microelectrodes in immature hippocampal slices and showed a lack of diffusion anisotropy; a tortuosity of about 1.39; and a volume fraction, alpha, of 0.41 +/- 0.01 (n = 10 slices). A reduction in osmolarity of -90 mOsm induced a decrease in alpha to 0.17 +/- 0.02 (n = 4 slices). The frequency of epileptiform activity elicited in 10-50 microM 4-AP-containing low-Mg2+ solution was increased under -90 mOsm and -40 mOsm hypoosmolar conditions by 39.9% +/- 8.1% (n = 16) and 24.1% +/- 4.8% (n = 10), respectively, whereas hyperosmolar solutions decreased the frequency. A -90-mOsm reduction in the osmolarity of low-Mg2+ solution induced epileptiform activity in nine of 19 slices. In summary, these results demonstrate that hypoosmolar conditions increased excitability and susceptibility to epileptiform activity in immature hippocampal slices, suggesting a functional role of the larger alpha in suppression of seizures.

We investigated the effect of L-type Ca2+ channel antagonists on the Ca2+ influx through voltage-gated Ca2+ channels in leech Retzius, Leydig, AP, AE, P, and N neurons. The efficacy of the antagonists was quantified by monitoring their effect on the increase in the intracellular free Ca2+ concentration ([Ca2+]i; measured by Fura-2) that was induced by depolarizing the cell membrane by raising the extracellular K+ concentration. This K+-induced [Ca2+]i increase was blocked by the phenylalkylamines verapamil, gallopamil, and devapamil, the benzothiazepine diltiazem, as well as by the 1,4-dihydropyridine nifedipine. The blocking effect of the three phenylalkylamines was similar, being most pronounced in P and N neurons and smaller in Leydig, Retzius, AP, and AE neurons. Contrastingly, diltiazem and nifedipine were similarly effective in the neurons investigated, whereby their efficacy was like that of the phenylalkylamines in Retzius, Leydig, AP, and AE neurons. Depending on cell type and blocking agent, the concentrations necessary to suppress the K+-induced [Ca2+]i increase by 50% were estimated to vary between 5 and 190 microM. At high concentrations, the phenylalkylamines and diltiazem by themselves caused a marked [Ca2+]i increase in Leydig, P, and N neurons, which is probably due to activation of the caffeine-sensitive ion channels present in the plasma membrane of these cells. Together with previous observations, the results indicate a distant relationship of the voltage-gated Ca2+ channels present in many if not all leech neurons to vertebrate L-type Ca2+ channels.

The role of 5-hydroxytryptamine (5-HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5-HT on the cytosolic free calcium concentration ([Ca2+]i) in leech neurons. As [Ca2+]i plays a pivotal role in numerous cellular processes, we investigated the effect of 5-HT on [Ca2+]i (measured by Fura-2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5-HT induced a [Ca2+]i increase which was predominantly due to Ca2+ influx since it was abolished in Ca2+-free solution. The 5-HT-induced Ca2+ influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage-dependent Ca2+ channels. In P and N1 neurons, the membrane depolarization was due to Na+ influx through cation channels coupled to 5-HT receptors, whereby the dose-dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5-HT receptor-coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage-dependent Ca2+ channels was evoked by 5-HT-triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5-HT had no effect on [Ca2+]i. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005

Changes in cell volume can be recorded by a potentiometric method based on ion-selective microelectrodes filled with the classical K + exchanger Corning 477317, which is very sensitive to large quaternary ammonium ions (PNAS 82 (1985) 6014: Electrochim. Acta 47 (2001) 309). We investigated the effect of hyperosmotic bathing solutions on cell volume, the intracellular concentrations of Na + , Cl - , and K + ([Na + ] i , [Cl - ] i , [K + ] i ) as well as on the membrane potential of leech Retzius neurones by using multi-barrelled ion-selective microelectrodes. We found that an increase in the extracellular NaCI concentration induced a shrinkage of the cells as well as an increase in [Na + ] i , [Cl - ] i and [K + ] i . Provided that the change in osmolality was moderate ( < +40%), cell shrinkage and the changes in [Na + ] i , [Cl - ] i and [K + ] i were proportional to the increase in the extracellular osmolality, i.e. the cells behaved like ideal osmometers. When the bath osmolality was increased more strongly, significant differences from the ideal behaviour were observed. In particular, the changes in [Na + ] i and [Cl - ] i were larger than expected from the changes in cell volume. The results show that leech neurones raise their intracellular osmolyte content in response to severe osmotic stress, thereby reducing changes in their cellular volume.

We investigated the cytosolic free Ca2+ concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic strength. Changing these parameters had no significant effect on [Ca2+]i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising the extracellular K+ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca2+]i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca2+]i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca2+]i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca2+]i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech Retzius neurons extracellular pH, but not intracellular pH, affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular side of the plasma membrane.

A potentiometric method to monitor cell volume changes in intact biological tissues is based on ion-selective microelectrodes filled with the classical K + exchanger Corning 477317, which is very sensitive to large quaternary ammonium ions. After introduction into a single cell these cations can be used as volume marker by relating their changes in concentration to changes in cell volume, provided that the ions are neither metabolized nor transported out of the cell. In this article we tested the applicability of this method in leech Retzius neurones. In addition to the cell volume, by using triple-barreled microelectrodes we also measured simultaneously the intracellular concentrations of physiologically relevant ion species as well as the membrane potential. We found that Retzius neurones possess an uptake mechanism which allowed for the intracellular accumulation of tetramethylammonium, tetraethylammonium or choline, simply by adding these ions to the bathing solution. In contrast, tetrapropylammonium was not taken up and therefore, we iontophoretically injected this ion species via conventional microelectrodes. The isoosmotic elevation of the extracellular K + concentration from 4 to 30 mM (Na + replaced by K + ) induced a swelling of the cells. The changes in the intracellular concentrations of K + and Na + suggest that cell swelling is primarily due to the uptake of K + , probably with Cl as counterion.

We investigated the effects of ATP (adenosine 5'-triphosphate) and derivatives on leech neuropile glial cells, focusing on exposed glial cells. ATP dose-dependently depolarized or hyperpolarized neuropile glial cells in situ as well as exposed neuropile glial cells. These potential shifts varied among cells and repetitive ATP application did not change their amplitude, duration or direction. In exposed neuropile glial cells, ATP most frequently induced a Na(+)-dependent depolarization and decreased the input resistance. The agonist potency ATP > ADP (adenosine 5'-diphosphate) > AMP (adenosine 5'-monophosphate) > adenosine indicates that P2 purinoceptors mediate this depolarization. The P2Y agonist 2-methylthio-ATP mimicked the ATP-induced depolarization, whereas the P2Y antagonist PPADS (pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid) reduced it. P2X agonists were without effect. Because the P1 antagonist 8-SPT (8-(p-sulphophenyl)-theophylline) also depressed ATP-induced depolarizations and some ATP-insensitive glial cells responded to adenosine, we suggest coexpression of metabotropic P2Y and P1 purinoceptors. The ATP-induced depolarization requires activation of Na(+) channels or nonselective cation channels, whereas the ATP-induced hyperpolarization indicates activation of K(+) channels. ATP also increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), that is independent of Ca(2+) influx but reflects intracellular Ca(2+) release possibly triggered by IP(3) formation. ADP and AMP also increased [Ca(2+)](i), but were less efficient than ATP; adenosine and 2-methylthio-ATP did not affect [Ca(2+)](i). In view of the mobilization of intracellular Ca(2+), ATP is clearly different from other leech neurotransmitters, because it enables intracellular Ca(2+) signaling without causing prominent changes in glial membrane potential. Thus disturbance of the extracellular microenvironment and the demand for metabolic energy are minimized.

1. The effect of kainate and other glutamatergic agonists on the membrane potential (Em), the intracellular Na+ activity (aNai), and the intracellular free Ca2+ concentration ([Ca2+]i) of identified leech neurons and neuropile glial cells was measured with conventional and ion-sensitive microelectrodes, as well as with the use of the iontophoretically injected fluorescent indicators sodium-binding benzofuran isophthalate and Fura-2. 2. In Retzius neurons, AE, L, 8, and 101 motoneurons, and in the unclassified 50 neurons (Leydig cells) and AP neurons, as well as in neuropile glial cells, bath application of 100 microM kainate evoked a marked membrane depolarization and an increase in aNai and [Ca2+]i. The kainate-induced aNai increase persisted in solutions with high Mg2+ concentration in which synaptic transmission is blocked. 3. A membrane depolarization as well as an increase in aNai and [Ca2+]i was also evoked by L-glutamate, quisqualate, and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). The agonist-induced [Ca2+]i increase was inhibited by 6,7-dinitroquinoxaline-2,3-dione (DNQX). 4. In Ca(2+)-free solution, the kainate-induced [Ca2+]i increase was abolished in the neurons and in neuropile glial cells, whereas membrane depolarization and aNai increase were unchanged. In Na(+)-free solution, kainate had no effect on Em, aNai, or [Ca2+]i in the neurons. 5. In the mechanosensory T, P, and N neurons, kainate induced considerably smaller membrane depolarizations than in the other neurons or in neuropile glial cells, and it had no significant effect on aNai or [Ca2+]i. 6. It is concluded that in leech segmental ganglia the majority of the neurons and the neuropile glial cells, but probably not the mechanosensory neurons, possess glutamate receptors of the AMPA-kainate type. In the neurons, the [Ca2+]i increase caused by glutamatergic agonists is due to Ca2+ influx through voltage-dependent Ca2+ channels that are activated by the agonist-induced membrane depolarization.

SUMMARYDuring periods of high activity neurons are expected to swell due to the uptake of Cl–. To find out whether leech Retzius neurons possess swelling-activated Cl– channels that facilitate Cl– efflux and, hence, volume recovery, we exposed the cells to hypotonic solutions. In hypotonic solutions, the cells slowly swelled but did not undergo a regulatory volume decrease. However, the cell volume increased less than predicted for an ideal osmometer, suggesting the action of a compensatory mechanism. The cell swelling was paralleled by a marked decrease in the input resistance as well as by the activation of a membrane current with a reversal potential close to the Cl–equilibrium potential. This current was substantially diminished by removing bath Cl–, by applying the Cl– channel blocker DIDS, or by treating the cells with the tubulin polymerization inhibitor colchicine. Furthermore, in the presence of colchicine or vinblastine, the cell swelling was substantially increased. It is concluded that leech Retzius neurons possess swelling-activated Cl–channels that require an intact microtubule system for activation. The channels may help to restore cell volume after periods of high neuronal activity.

By using electrophysiological and microfluorimetric methods, we found that leech Retzius neurons swell after inhibition of the Na(+)-K(+) pump by the cardiac glycoside ouabain. To explore the mechanism of this swelling, we measured the effect of ouabain on [Na(+)](i), [K(+)](i), and [Cl(-)](i), as well as on the membrane potential, by applying triple-barrelled ion-sensitive microelectrodes. As shown previously, ouabain induced a marked [Na(+)](i) increase, a [K(+)](i) decrease, and a membrane depolarization, and it also evoked an increase in [Cl(-)](i). The analysis of the data revealed a net uptake of NaCl, which quantitatively explained the ouabain-induced cell swelling. In the absence of extracellular Na(+) or Cl(-), NaCl uptake was excluded, and the cell volume remained unaffected. Likewise, NaCl uptake and, hence, cell swelling did not occur when the Na(+)-K(+) pump was inhibited by omitting bath K(+). Also, in K(+)-free solution, [Na(+)](i) increased and [K(+)](i) dropped, but [Cl(-)](i) slightly decreased, and after an initial, small membrane depolarization, the cells hyperpolarized for a prolonged period. It is concluded that the ouabain-induced NaCl uptake is caused by the depolarization of the plasma membrane, which augments the inwardly directed electrochemical Cl(-) gradient.

In leech P neurons the inhibition of the Na(+)-K(+) pump by ouabain or omission of bath K(+) leaves the membrane potential unaffected for a prolonged period or even induces a marked membrane hyperpolarization, although the concentration gradients for K(+) and Na(+) are attenuated substantially. As shown previously, this stabilization of the membrane potential is caused by an increase in the K(+) conductance of the plasma membrane, which compensates for the reduction of the K(+) gradient. The data presented here strongly suggest that the increased K(+) conductance is due to Na(+)-activated K(+) (K(Na)) channels. Specifically, an increase in the cytosolic Na(+) concentration ([Na(+)](i)) was paralleled by a membrane hyperpolarization, a decrease in the input resistance (R(in)) of the cells, and by the occurrence of an outwardly directed membrane current. The relationship between R(in) and [Na(+)](i) followed a simple model in which the R(in) decrease was attributed to K(+) channels that are activated by the binding of three Na(+) ions, with half-maximal activation at [Na(+)](i) between 45 and 70 mM. At maximum channel activation, R(in) was reduced by more than 90%, suggesting a significant contribution of the K(Na) channels to the physiological functioning of the cells, although evidence for such a contribution is still lacking. Injection experiments showed that the K(Na) channels in leech P neurons are also activated by Li(+).

Because changes in cell volume might disturb the normal function of animal cells, most cells are endowed with volume-regulating mechanisms. Experimentally induced changes in cell volume are often paralleled by changes in the membrane potential, which might affect a variety of transport processes across the cell membrane and, in turn, volume regulation [1, 2]. We have shown previously that multi-barrelled ion-selective microelectrodes are useful tools for measurement of cell volume and the intracellular concentrations of ions that might be relevant to volume regulation [1]. To investigate whether voltage-dependent transport processes are involved in cell-volume regulation we combined the potentiometric technique of ion-selective microelectrodes with the voltage-clamp technique. This combination enables simultaneous recording of cell volume, relevant intracellular ion concentrations, and ion currents across the cell membrane at a fixed membrane potential.