Ultrastructure of Aplysia neurons having different degrees of light sensitivity.

The relationship between ultrastructure and photosensitivity of pigmented neurons of the abdominal ganglion of Aplysia californica was investigated using electron microscopy and electrophysiological methods. Four identified neurons of similar light microscopic appearance were examined; two are photoresponsive and two are not. Illumination hyperpolarizes both responsive neurons. One of them, R2, requires roughly 100 times greater light intensities than does the other, the ventral photoresponsive neuron (VPN), for similar responses. Two neurons lying adjacent to VPN and similar in appearance to VPN do not have measurable electrophysiological responses to even the highest light intensities. All four neurons contained lipochondria, pigmented organelles associated with the light response. Therefore the presence of these organelles is not the only requirement for light sensitivity in these neurons. Illumination appeared to increase the number of membranous lipochondria in both R2 and the ventral neurons, but only in R2 was this increase significant. Factors such as the concentration of lipochondria near the plasma membrane may affect quantitative aspects of the light response, but in the insensitive cells the lipochondria are apparently uncoupled from other factors required for the light response.     

Isolation of pigmented granules involved in extra-retinal photoreception in Aplysia californica neurons

Many neurons in the ganglia of Aplysia california contain pigmented, membrane-bound granules (lipochondria), which are thought to mediate the light response of some of the neurons, including the giant cell of the abdominal ganglion. A method of isolating the lipochondria by centrifugation of ganglia homogenates has now been developed. Electron microscopy was used to demonstrate that most of the lipochondria remain morphologically intact. As shown by X-ray microanalysis, isolated lipochondria contain the same elements, including calcium, as do lipochondria in intact giant cells. The calcium can be released into the medium by treatment of the organelles with the Ca²⁺ ionophore A23187. It appears that the lipochondria of Aplysia ganglia are similar in their morphology, elemental content and susceptibility to the ionophore. Two pigments were isolated from the lipochondria, and chromatography and spectrophotometric studies indicated that they are β-carotene and a “retinol-like” compound.     

Lipochondria and the light response of Aplysia giant neurons

The ultrastructure, absorbance, and elemental content of lipochondria present in the cytoplasm of Aplysia giant neurons have been investigated before and after 30–1,200 sec doses of white light at intensities which produce saturated light responses. The effects of exposure to the calcium ionophore A-23187 and to EGTA were also examined. The lipochondria of nonilluminated neurons are membrane-bound, and contain lipids, protein, Na, K, Mg, Ca, Si, Cl, Br, P, and a pigment which is probably β-carotene. The cytoplasm appeared to have little pigment. When neurons were illuminated for 20 min, 60–70% of the lipochondria showed marked ultrastructural alterations, the most notable being the appearance of membranous material. Earlier changes which occur after 30 sec of illumination include the appearance of paracrystalline arrays and mottling. Less than 10% of lipochondria in nonilluminated neurons have a similar appearance. These effects were greatly enhanced in illuminated neurons exposed to the calcium ionophore or EGTA. In nonilluminated neurons, the ionophore also produced ultrastructural changes. In frozen specimens, the calcium content of the most electron dense lipochondria of illuminated neurons was reduced. Other elements which were counted were also reduced. The lipochondria are the main intracellular site of photopigment. They may also act as an intracellular source for calcium which, as the accompanying paper indicated, may mediate phototransduction in Aplysia neurons.     

Lipochondria and the light response of Aplysia giant neurons,.

The ultrastructure, absorbance, and elemental content of lipochondria present in the cytoplasm of Aplysia giant neurons have been investigated before and after 30-1,200 sec doses of white light at intensities which produce saturated light responses. The effects of exposure to the calcium ionophore A-23187 and to EGTA were also examined. The lipochondria of nonilluminated neurons are membrane-bound, and contain lipids, protein, Na, K, Mg Ca, Si, Cl, Br, P, and a pigment which is probably beta-carotene. The cytoplasm appeared to have little pigment. When neurons were illuminated for 20 min, 60-70% of the lipochondria showed marked ultrastructural alterations, the most notable being the appearance of membranous material. Earlier changes which occur after 30 sec of illumination include the appearance of paracrystalline arrays and mottling. Less than 10% of lipochondria in nonilluminated neurons have a similar appearance. These effects were greatly enhanced in illuminated neurons exposed to the calcium ionophore or EGTA. In nonilluminated neurons, the ionophore also produced ultrastructural changes. In frozen specimens, the calcium content of the most electron dense lipochondria of illuminated neurons was reduced. Other elements which were counted were also reduced. The lipochondria are the main intracellular site of photopigment. They may also act as an intracellular source for calcium which, as the accompanying paper indicated, may mediate phototransduction in Aplysia neurons.     

Light-sensitive voltage responses in the neurons of the cerebral ganglion of Ciona savignyi (Chordata: Ascidiacea)

Light-responsive behaviors such as siphon contraction (1), phototropism (2), and gamete release (3, 4) have been described in several ascidian species. The pigmented spots around the siphon openers (5), the epithelial cells of the sperm duct (6, 7), and the cerebral ganglion (8) have been suggested to be the photoreceptor candidates underlying these behaviors. However, these arguments have not yet been settled because no direct electrophysiological recordings of light-induced receptor potentials have been reported. In this study, we focused on the cerebral ganglion and performed intracellular recordings from the neurons in the ventral side of the cerebral ganglion in an isolated in vitro preparation of the neural complex in Ciona savignyi. We found that 24% (n = 115) of the recorded neurons showed various types of voltage responses to light stimuli. Almost all (27/28) of the recorded voltage responses were "on" responses that included hyperpolarizing and depolarizing responses and could be categorized into five types, except for a complex response recorded in one cell; the remaining one (1/28) was a depolarizing "off" response. This is the first report of electrophysiological recordings of light-sensitive voltage responses from ascidian cerebral ganglion neurons.     

Spectral characterization of the neuronal pigments of Aplysia juliana

Spectral analysis at liquid N₂ temperature of the circumesophageal ganglia of Aplysia juliana showed that carotenoids and a hemoglobin-like pigment are contained in concentrations of approx. 25 and 3 μM, respectively, in the whole ganglia. Microspectrophotometrical measurements of Aplysia neurons indicated that the carotenoids reside on lipochondria in a concentration of approx. 38 mM. In addition to lipochondria, two types of pigmented particulate having absorption maxima at about 512 and 525 nm, respectively, were found in the neurons. The neuronal carotenoids consist of violaxanthin, β-carotene and one minor component; among them the first occupies approx. 77% of total carotenoids. Two principal absorption maxima of the carotenoids, when existing in both ganglial homogenates and Triton X-100 extracts, show a red shift of 10 nm compared with those of free pigments in hexan. The red shift may be interpreted as due to the solvation of the carotenoids by surrounding lipids     

Age-dependent anatomical changes in an identified neuron in the cns of Aplysia californica

Neurons of Aplysia californica are naturally pigmented and the pigment accumulates with age. In the present study the pigment was examined in the same neuron from Aplysia of three postmetamorphic ages: young, sexually mature, and old. The large central neuron, R₂, was examined by light and electron microscopy to determine if the pigment possessed properties similar to lipofuscin pigment seen in aging mammalian neurons. We used the same microscopic techniques that demonstrate the presence of lipofuscin in mammalian neurons. Light microscopic studies demonstrated a regional correlation between autofluorescence, staining with Sudan Black, and the naturally occurring pigment in old R₂s. Electron microscopic studies revealed the presence of large vacuolated and lamellated membrane-bound bodies in the peripheral cytoplasm of old R₂s, similar to those found in mammalian neurons. The bodies were located in the same region in which autofluorescence and Sudan Black staining were observed. Although the naturally occurring pigment accumulates with age, it acquires characteristics of lipofuscin pigment in the neurons of older sexually mature animals. The presence of these pigment characteristics can be used as an index of aging in Aplysia neurons as they are in mammalian neurons.     

Central organization of common inhibitory motoneurons in the locust: role of afferent signals from leg mechanoreceptors

Intracellular recordings of mesothoracic common inhibitory neurons (CI₁, CI₂ and CI₃) were made while tactile hairs of the middle legs of locusts (Locusta migratoria) were mechanically stimulated. Generally the three common inhibitory neurons were excited by stimulation of tactile hairs on the ventral and dorsal surface of femur and tibia. The response pattern of all three CI neurons was similar suggesting that they work as a functional unit. Touching hairs on the dorsal surface of tibia and tarsus in some cases led to inhibition of CIs. The connection between sensory cells of tactile hairs and common inhibitory neurons is polysynaptic.To identify interneurons which mediate afferent signals, simultaneous intracellular recordings from CIs and interneurons were made. Different spiking interneurons were identified which made excitatory or inhibitory monosynaptic connections with CIs. Interneurons with inhibitory input to CIs belonged to the ventral midline group of spiking local interneurons. Behavioral and electrophysiological results indicate that reflex movements of the leg are accompanied by activity of CI neurons. Further it appears that CI activity is inhibited when reflex movements of the leg are actively suppressed by the animal.Abbreviations CI common inhibitor - IN interneuron - LY Lucifer Yellow     

The Effect of Photosynthetic Enhancement on Photorespiration in Sinapis alba

High external concentrations of potassium were found to promote light-induced growth of cucumber cotyledons similarly to the effects previously observed for the growth induced by cytokinins. At 40 mM KCl, the response to white light was 3.6 times greater than in the absence of KCl. The promotive effect of calcium on the growth induced by cytokinin was not observed for light. In 40 mM KCl and 10 mM CaCl₂, the responses to light and cytokinin were similar and additive. Both near-red and far-red light induced growth at low intensities. The response to white light at low intensities was sharply increased with higher intensity up to 24 μE m^-2 s^-1 and only slightly increased above that level. Abscisic acid was found to inhibit strongly the responses to light and cytokinin. The inhibition was greater in the presence of KCl than in its absence and thus abscisic acid appeared to inhibit primarily by its interference with potassium uptake. Kinetic analysis found the light response promoted by potassium to be inhibited competitively by abscisic acid, with the light response having a K_m of 34 mM KCl and a V_max of 115 mg/cotyledon × 4 days. The inhibition of the cytokinin response by abscisic acid was noncompetitive in relation to potassium, having a K_m of 1 mM KCl and a V_max of 50 mg/cotyledon × 4 days. It is suggested that cytokinin, light and abscisic acid have primary properties affecting membrane permeability and that their interaction with potassium is an explanation of many similarities between light and cytokinin responses and their inhibition by abscisic acid.     

Physiological Characteristics of Photosynthesis and Respiration in Stems of Populus tremuloides Michx

The physiological responses of 6- to 8-year-old aspen (Populus tremuloides Michx.) stems to temperature, light, and CO₂ concentration were investigated in the field throughout the year using infrared CO₂ analysis. Light response studies showed that the rate of gross photosynthesis was linear from 0 to 400 ft-c (0 to 1.6 mw/cm² of 400-700 nm) with light saturation being reached between 800 to 1400 ft-c (3.2 to 5.6 mw/cm² of 400-700 nm). At this light intensity, the respiratory CO₂ loss was reduced to 10 to 15% of dark rates. Net photosynthetic CO₂ uptake was not observed even at intensities as high as 3400 ft-c (13.6 mw/cm² of 400-700 nm). The light response curve was similar for both winter and summer stems.     

Effects of increased intracellular pH-buffering capacity on the light response of Limulus ventral photoreceptor

Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A I M solution of either a zwitter-ionic pH buffer or a weakly-buffering control substance was pressure injected through a micropipette into a ventral photoreceptor cell. To estimate the amount injected, ³⁵SO₄ was included in the solution. Membrane currents induced by light flashes were measured by a voltage-clamp technique. The buffer-filled micropipette passed current and a 3 M KCl filled micropipette monitored membrane voltage. The sensitivity (peak light-induced current/stimulus energy) was measured, after dark adaptation, before and after injection. Injections of buffers, pH 6.3–7.2, to intracellular concentrations of at least 40–200 mM produced only a small mean decrease in sensitivity, approximately equal to that caused by injections of control substances. Excitation, therefore, apparently is not mediated by a change in intracellular pH. Buffers with pH values 5.4–8.4 were also injected. The time to peak of the response depended on pH, being shortened by up to 20% at pH values below 7.7 and lengthened at higher pH values. The time to peak of the response appeared to be shortened by an increase in intracellular pH-buffering capacity even when there was no change in intracellular pH.     

Projection pattern of sensory neurons in the central nervous system of a homeotic mutation of the moth Manduca sexta

Octopod (Octo) is a mutation of the moth Manduca sexta, which transforms the first abdominal segment (A1) in the anterior direction. Mutant animals are characterized by the appearance of homeotic thoracic-like legs on A1. We exploited this mutation to determine what rules might be used in specifying the fates of sensory neurons located on the body surface of larval Manduca. Mechanical stimulation of homeotic leg sensilla did not cause reflexive movements of the homeotic legs, but elicited responses similar to those observed following stimulation of ventral A1 body wall hairs. Intracellular recordings demonstrated that several of the motoneurons in the A1 ganglion received inputs from the homeotic sensory hairs. The responses of these motoneurons to stimulation of homeotic sensilla resembled their responses to stimulation of ventral body wall sensilla. Cobalt fills revealed that the mutation transformed the segmental projection pattern of only the sensory neurons located on the ventral surface of A1, resulting in a greater number with intersegmental projection patterns typical of sensory neurons found on the thoracic body wall. Many of the sensory neurons on the homeotic legs had intersegmental projection patterns typical of abdominal sensory neurons: an anteriorly directed projection terminating in the third thoracic ganglion (T3). Once this projection reached T3, however, it mimicked the projections of the thoracic leg sensory neurons. These results demonstrate that the same rules are not used in the establishment of the intersegmental and leg-specific projection patterns. Segmental identity influences the intersegmental projection pattern of the sensory neurons of Manduca, whereas the leg-specific projections are consistent with a role for positional information in determining their pattern. © 1995 John Wiley & Sons, Inc.     

Influences of superior laryngeal afferent stimulation on expiratory activity in cats

The effects of superior laryngeal nerve (SLN) stimulation on the activity of the expiratory muscles and medullary expiration-related (ER) neurons were investigated in 24 pentobarbital-anesthetized cats. In some experiments the animals were also paralyzed and artificially ventilated. Sustained tetanic stimulation of SLN consistently caused an apneic response associated with the appearance of tonic CO2-dependent activity in the expiratory muscles and in ER neurons located in the caudal ventral respiratory group (VRG) and the B?tzinger complex. Single shocks or brief tetani at the same stimulation intensities failed to evoke excitatory responses in the expiratory muscles and in the vast majority of ER neurons tested. At higher stimulation strengths, single shocks or short tetani elicited excitatory responses in the expiratory muscles (20- to 35-ms latency) and in the majority of ER neurons of the caudal VRG (7.5- to 15.5-ms latency). These responses were obtained only during the expiratory phase and proved to be CO2 independent. On the contrary, only inhibitory responses were evoked in the activity of B?tzinger complex neurons. The observed tonic expiratory activity most likely represents a disinhibition phenomenon due to the suppression of inspiratory activity; activation of expiratory muscles at higher stimulation intensities appears to be a polysynaptic reflex mediated by ER neurons of the caudal VRG but not by B?tzinger complex neurons.     

Dopamine and serotonin effects on neurons of dorsal root ganglion isolated from rats

The effects of 1×10^–8–1×10^–5 M dopamine (DA) and serotonin (HT) on membrane potential, input resistance (R_M), and action potential (AP) when added to the superfusing fluid for 0.5 min were investigated in perfused dorsal root ganglia (DRG) neurons isolated from 30–36-day old rats during experiments using intracellular recording techniques. Application of DA induced reversible changes in membrane potential in 48 out of 52 test cells as compared with 38 out of 44 for HT. Distribution of different patterns of response to DA and HT was similar: depolarization was recorded in 64.6 and 73.7% and hyperpolarization in 16.7 and 15.8%; two-stage response occurred in 18.7 and 10.5% of responding cells, respectively. Both monoamines induced reversible change in the AP and R_M pattern in a number of cells. Depolarization was accompanied by a decline and hyperpolarization by a rise in R_M. Both substances were found to affect mainly those neurons with electrophysiological properties characteristic of small cells. The possibility of afferent spike train modulation at the level of primary sensory neurons is suggested.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian USSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 5, pp. 644–651, September–October, 1989.     

Predicting ecosystem carbon balance in a warming Arctic: the importance of long‐term thermal acclimation potential and inhibitory effects of light on respiration

The carbon balance of Arctic ecosystems is particularly sensitive to global environmental change. Leaf respiration (R), a temperature‐dependent key process in determining the carbon balance, is not well‐understood in Arctic plants. The potential for plants to acclimate to warmer conditions could strongly impact future global carbon balance. Two key unanswered questions are (1) whether short‐term temperature responses can predict long‐term respiratory responses to growth in elevated temperatures and (2) to what extent the constant daylight conditions of the Arctic growing season inhibit leaf respiration. In two dominant Arctic species E riophorum vaginatum (tussock grass) and B etula nana (woody shrub), we assessed the extent of respiratory inhibition in the light (R _L/R _D), respiratory response to short‐term temperature change, and respiratory acclimation to long‐term warming treatments. We found that R of both species is strongly inhibited by light (averaging 35% across all measurement temperatures). In E . vaginatum both R _L and R _D acclimated to the long‐term warming treatment, reducing the magnitude of respiratory response relative to the short‐term response to temperature increase. In B . nana, both R _L and R _D responded to short‐term temperature increase but showed no acclimation to the long‐term warming. The ability to predict plant respiratory response to global warming with short‐term temperature responses will depend on species‐specific acclimation potential and the differential response of R _L and R _D to temperature. With projected woody shrub encroachment in Arctic tundra and continued warming, changing species dominance between these two functional groups, may impact ecosystem respiratory response and carbon balance.     

DNase and RNase Responses of Petunia × hybrida During Transition to Flowering as Affected by Light Treatments

The responses of DNase and RNase isoforms and their specific activities following transition to flowering (1 to 6 weeks) were examined in Petunia × hybrida under different light conditions. Petunia × hybrida plants formed flower buds at the 4^th week in the case of high light and at the 6^th week in the far-red light treatment, while no flower bud formation was observed upon red light and control light treatments. The DNase and RNase activities decreased from the 1^st to the 6^th week during transition to flowering. Native-PAGE analysis revealed the appearance of one DNase (D₁) and seven RNase (R₁ - R₇) isoforms in all light treatments. It is assumed that the progress of the flowering could be related to the disappearance or reduction of D₁ DNase band intensity and disappearance of R₁, R₂ and R₇ RNase isoforms. Consequently, these isoforms could be used as potent biochemical markers of flower bud formation under light intensity as well as light quality treatments.     

Phototaxis in a dinoflagellate: Action spectra as evidence for a two-pigment system

Summary Action spectra were determined in the UV region of the spectrum for the first phase of the phototactic response (stop response) and for the phytochrome pigment associated with this response in the dinoflagellate Gyrodinium dorsum Kofoid. Differences between these action spectra indicate the participation of two pigments in phototaxis. Following R (620 nm) irradiation of the phytochrome, the stop response maxima occur at 470 and 280-nm; after FR irradiation they shift to 490 and 300–310 nm. These maxima suggest that the photoreceptor pigment for phototaxis is a carotenoprotein. The action spectrum shift following the different phytochrome conversions may represent a trans to cis isomer change by the carotenoid. The absorption maximum of P_R in the UV appears to be at 320 nm, which is consistent with the shift of the R absorption maximum to shorter wavelengths (620 nm) as compared to higher plants. The P_FR absorption maximum appears as a broad band between 360 and 390 nm. Comparison of P_R to P_FR conversions by different intensities of 620-nm and 320-nm light indicates that at lower intensities the logarithm of the threshold for the stop response is inversely proportional to the logarithm of the intensity of the sensitizing light. The ratio of response activation by R and UV light is about 4:1.Abbreviations FR far-red - R red - P_FR far-red-absorbing form of phytochrome - P_R red-absorbing form of phytochrome - UV ultraviolet     

Conventional measures of intrinsic excitability are poor estimators of neuronal activity under realistic synaptic inputs

Activity-dependent regulation of intrinsic excitability has been shown to greatly contribute to the overall plasticity of neuronal circuits. Such neuroadaptations are commonly investigated in patch clamp experiments using current step stimulation and the resulting input-output functions are analyzed to quantify alterations in intrinsic excitability. However, it is rarely addressed, how such changes translate to the function of neurons when they operate under natural synaptic inputs. Still, it is reasonable to expect that a strong correlation and near proportional relationship exist between static firing responses and those evoked by synaptic drive. We challenge this view by performing a high-yield electrophysiological analysis of cultured mouse hippocampal neurons using both standard protocols and simulated synaptic inputs via dynamic clamp. We find that under these conditions the neurons exhibit vastly different firing responses with surprisingly weak correlation between static and dynamic firing intensities. These contrasting responses are regulated by two intrinsic K-currents mediated by Kv1 and K_ir channels, respectively. Pharmacological manipulation of the K-currents produces differential regulation of the firing output of neurons. Static firing responses are greatly increased in stuttering type neurons under blocking their Kv1 channels, while the synaptic responses of the same neurons are less affected. Pharmacological blocking of K_ir-channels in delayed firing type neurons, on the other hand, exhibit the opposite effects. Our subsequent computational model simulations confirm the findings in the electrophysiological experiments and also show that adaptive changes in the kinetic properties of such currents can even produce paradoxical regulation of the firing output.     

Ultrastructure of the retinal pigmented epithelium of light- and dark-adapted young,pigmented, and mature silver eels,Anguilla anguilla (Pisces,Teleostei)

Summary Our ultrastructural study of the retinal pigmented epithelium (RPE) of young, pigmented, and mature silver eels, (Anguilla anguilla), revealed the presence of retinomotor responses in the RPE of both growth stages. Furthermore, while the overall fine structure of the RPE is similar to that of other vertebrates, specific structures were also found. These include various forms of lipid droplets, intercellular membranous whorls, multitubular bodies, macrophages, melanolysosomes, and myeloid bodies (large and numerous in young eels). Structural variations in cellular organelles of the RPE according to different adaptive states (light, dark) and growth stages (young, mature) of the animal are discussed with respect to their significance in its complex life cycle.     

Prefrontal Cortex Haemodynamics and Affective Responses during Exercise: A Multi-Channel Near Infrared Spectroscopy Study

The dose-response effects of the intensity of exercise upon the potential regulation (through top-down processes) of affective (pleasure-displeasure) responses in the prefrontal cortex during an incremental exercise protocol have not been explored. This study examined the functional capacity of the prefrontal cortex (reflected by haemodynamics using near infrared spectroscopy) and affective responses during exercise at different intensities. Participants completed an incremental cycling exercise test to exhaustion. Changes (Δ) in oxygenation (O₂Hb), deoxygenation (HHb), blood volume (tHb) and haemoglobin difference (HbDiff) were measured from bilateral dorsal and ventral prefrontal areas. Affective responses were measured every minute during exercise. Data were extracted at intensities standardised to: below ventilatory threshold, at ventilatory threshold, respiratory compensation point and the end of exercise. During exercise at intensities from ventilatory threshold to respiratory compensation point, ΔO₂Hb, ΔHbDiff and ΔtHb were greater in mostly ventral than dorsal regions. From the respiratory compensation point to the end of exercise, ΔO₂Hb remained stable and ΔHbDiff declined in dorsal regions. As the intensity increased above the ventilatory threshold, inverse associations between affective responses and oxygenation in (a) all regions of the left hemisphere and (b) lateral (dorsal and ventral) regions followed by the midline (ventral) region in the right hemisphere were observed. Differential activation patterns occur within the prefrontal cortex and are associated with affective responses during cycling exercise.