Extracellular ATP in micromolar concentrations evokes a transient elevation in intracellular free Ca2+ concentration ([Ca2+]i), which arises primarily from a release of Ca2+ from intracellular stores in rat brown adipocytes. We investigated the mechanisms underlying this transient nature of [Ca2+]i elevation during exposure to ATP by using fura-2 fluorescence measurements together with the P2 receptor antagonists pyridoxal-phosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS) and suramin. Extracellular ATP (10 µM) almost completely depressed the thapsigargin (100 nM)-evoked [Ca2+]i elevation mediated through storeoperated Ca2+ entry. The inhibitory effect of ATP was antagonized by PPADS with IC50 of 0.7 µM. In the presence of PPADS at concentrations of more than 5 µM, the ATP-induced [Ca2+]i elevation became sustained during the entire duration of the agonist application, although the magnitude of the sustained [Ca2+]i elevation was reduced in a concentration-dependent manner by PPADS with an IC50 of 200 µM. In contrast, the ATPinduced [Ca2+]i elevation was blocked by suramin in a concentration range similar to that required to antagonize the inhibitory effect of ATP on the store-operated pathway. These results suggest that the [Ca2+]i responses to extracellular ATP in rat brown adipocytes are mediated through the activation of at least two distinct P2 receptors exhibiting different sensitivities to PPADS but similar sensitivities to suramin. Extracellular ATP stimulates the PPADS-resistant P2 receptor to mobilize intracellular Ca2+ stores, which is probably followed by the activation of store-operated Ca2+ entry. Extracellular ATP, however, would inhibit this Ca2+ entry process through the stimulation of the PPADS-sensitive P2-receptor, which may underlie the transient nature of [Ca2+]i elevation in response to extracellular ATP.

We examined whether protein kinase C(PKC) modulates the transport systems involved in lactate movements across the plasma membranes of rat jejunum. In vitro phosphorylated membrane vesicles were used to perform uptake studies, the results of which suggested that PKC activation exerts an inhibitory effect on basolateral H+–lactate symport, as well as on apical Na+–glucose cotransport. The specificity of the response to PKC was confirmed by using staurosporine, chelerythrine or 4-α-PMA. Experiments performed using the whole tissue incubated in vitro confirmed the reduction of lactate transport elicited by PKC and gave evidence for an associated inhibition of fluid transport. Na+,K+-ATPase activity seems to be unaffected by the kinase and inhibited by Ca2+. Taken together, our results suggest that the overall action of PKC results from the simultananeous modulation of multiple pathways, targeted to a reduction of both lactate and bicarbonate transports without altering cell pH homeostasis.

The aim of the present study was to determine the influence on renal sympathetic nerve activity of the different chemically coded neuronal phenotypes that project from the paraventricular nucleus (PVN) to the spinal cord. Experiments were carried out on male Wistar rats anaesthetised with chloralose and urethane. Changes in renal sympathetic nerve activity were measured following activation of neurones in the PVN with D,L-homocysteic acid (100 nl, 200 mM), before and following intrathecal application of glutamate, vasopressin, oxytocin, dopamine and their receptor antagonists. Excitatory and inhibitory effects on renal sympathetic nerve activity were elicited by PVN stimulation. PVN excitatory effects were mimicked by intrathecal administration of glutamate and vasopressin and selectively antagonised by intrathecal administration of kynurenic acid and a V1a receptor antagonist, respectively. A low dose of dopamine increased renal sympathetic activity and this was selectively antagonised by haloperidol; however, the latter was without effect on PVN excitatory responses. A high dose of dopamine decreased renal sympathetic nerve activity and this was selectively blocked by a D1 dopamine receptor antagonist (SCH 23390), which also antagonised a minority of inhibitory responses obtained from the caudal extension of the PVN. Oxytocin also had two actions: in 5 rats it inhibited and in 10 rats it increased renal sympathetic nerve activity, both actions being blocked selectively by oxytocin receptor antagonists. Neither of the PVN effects on renal sympathetic nerve activity appeared to be dependent on oxytocin pathways. Tests with intrathecal administration of bicuculline showed that PVN inhibition of renal sympathetic nerve activity was not dependent on spinal GABAA receptor activation. The results show that PVN-induced excitation of sympathetic activity to the kidney is mainly mediated by glutamate or vasopressin neurones whereas dopamine via D1 receptors may mediate some of the PVN inhibitory effects.

Three experiments were performed to see whether cardiac neurons contribute to the intrinsic control of heart rate in right atria of adult rats. The intrinsic heart rate response (IRR) was examined by raising right atrial pressure from 2 to 8 mmHg for 3 min. In isolated preparations of the right atrium, the IRR was not significantly altered by the addition of either 1 mM atropine (n = 6; control +19 ± 3 min−1; atropine +18 ± 3 min−1 (mean ± S.E.M.)) or 1 mM propranolol (n = 5; control +22 ± 4 min−1; propranolol +21 ± 3 min−1). Tetrodotoxin (0.5 µM) had no effect on the IRR (n = 6; control +37 ± 5 min−1; tetrodotoxin 38 ± 5 min−1). In another experiment, 2-day-old rat pups were injected with capsaicin (50 mg kg−1; treated) or with vehicle (control). There was no difference in the IRR of right atrial preparations taken from control and treated animals after they reached adulthood (control (n = 7) and treated (n = 8): +30 ± 4 and +32± 4 min−1). The influence of right atrial pressure on the efficacy of vagal stimulation was examined. The rate response to vagal stimulation was reduced similarly in control and treated preparations when pressure was elevated from 2 to 4 mmHg (control and treated: −34 ± 5% and −33 ± 3%). The effectiveness of the capsaicin treatment was confirmed by the depletion of substance P-immunoreactive nerve fibres in cardiac tissues. Together, these results strongly suggest that cardiac neurons are not involved in intrinsic heart rate control.

Primary thoracic blast injury causes a triad of bradycardia, hypotension and apnoea mediated in part via a vagal reflex. Blast casualties may also suffer blood loss, and the response to progressive simple haemorrhage is biphasic: an initial tachycardia followed by a vagally mediated reflex bradycardia which can be attenuated by μ opioid agonists. The aims of this study were to determine the effects of thoracic blast injury on the response to subsequent haemorrhage, and the effects of morphine, administered after blast, on the response to blood loss. Male Wistar rats, terminally anaesthetised with alphadolone–alphaxolone (19–21 mg kg−1 h−1 I.V.), were allocated randomly to one of three groups: Group I, sham blast; Group II, thoracic blast; Group III, thoracic blast plus morphine (0.5 mg kg−1 I.V. given 5 min after blast). Blast (Groups II and III) resulted in significant (P<0.05, ANOVA) bradycardia, hypotension and apnoea. Sham blast (Group I) had no effect. Ten minutes later, haemorrhage (40% of the estimated total blood volume (BV)) in Group I produced a biphasic response comprising a tachycardia followed by a peak bradycardia after the loss of 33% BV. Arterial blood pressure did not fall significantly until the loss of 13.3% BV. In Group II the haemorrhage-induced tachycardia was absent and the bradycardia was augmented: peak bradycardia was seen after the loss of 23% BV. Mean arterial blood pressure (MBP) began to fall as soon as haemorrhage commenced and was significant after the loss of 10% BV. Morphine (Group III) prevented the haemorrhage-induced bradycardia and delayed the significant fall in MBP until the loss of 30% BV. It is concluded that the response to thoracic blast injury augments the depressor response to haemorrhage while morphine attenuates this response.

The length of cooperative units along the thin filament of rabbit psoas single muscle fibres was determined by reducing filament length by treatment with the thin filament severing protein, gelsolin, in the presence of Ca2+ and 2,3-butanedione 2-monoxime (BDM). The average time for 50% reduction in isometric tension was 6.7 min at 22°C. The pCa–tension relationship was measured at 22°C, pH 7.00 and ionic strength 200 mM, and the data were fitted to the Hill equation to determine the half-saturation point (K) and the cooperativity (n). Our results demonstrate that the cooperativity does not change much when the remaining isometric tension was in the range 20–100%. The cooperativity quickly diminished when the remaining tension was reduced to less than 20%. Our results further demonstrate that the change in the pK value was minimal and averaged 0.075 (less Ca2+ sensitive) as the thin filament length was reduced. We infer from the first observation that the thin filament cooperativity extends up to 0.2 μm, which includes the maximum of about four basic cooperative units consisting of seven actin molecules, one tropomyosin dimer and one troponin complex. We infer from the second observation that the Ca2+ sensitivity is slightly reduced by removal of the cooperative interaction between neighbouring cooperative units.

The purpose of the study was to investigate whether jaw reflexes evoked by selective stimulation of periodontal ligament mechanoreceptors are susceptible to modulation by remote noxious stimulation. Experiments were performed on 10 volunteer subjects. Skin surface recordings were made from the jaw-closing masseter muscle. The subjects activated the muscle to approximately 10% of maximum by biting on a rubber impression of their molar teeth while they received visual feedback of the electromyogram (EMG) of the muscle. Reflexes were produced by the application of gentle mechanical stimuli to an upper central incisor tooth. The stimuli were in the form of ‘ramp and hold’ forces with a 5 ms rise-time and a 1.5 N plateau which lasted 350ms. The resulting reflexes were recorded both under control conditions and while the subjects received a remote noxious stimulus (immersion of a hand in water at 3°C). In all 10 subjects, the stimuli produced a single period of inhibition of masseteric activity (latency, 12.8±0.4 ms; duration, 18.1±1.3 ms; means ± S.E.M.), which was usually followed by a period of increased masseteric activity. The period of inhibition constituted a downward wave in full-wave rectified, averaged signals. The integrals of such waves were significantly smaller (by 17±6.5 %; P = 0.027; Student's t test) when the reflex was evoked during remote noxious stimulation rather than under control conditions. As such reflexes are believed to play a modulatory role during normal oral function, this finding may be relevant to disorders of mastication associated with pain.

Multiphoton microscopy is based on the simultaneous absorption of two photons emitted by a pulsed infrared laser source. In this technique, the excitation is restricted to a very small focus and thus results in optical sectioning a priori without the need of a confocal aperture. Multiphoton microscopy was introduced in live cell imaging as an alternative to confocal microscopy due to its superior qualities, such as the deep penetration depth, the reduced photodamage and the lack of out-of-focus bleaching. However, during the past years, examinations revealed severe limitations to the initial expectations. In the focal plane, photodamage and photobleaching can be worse than in single photon microscopy. However, studies showed that with low excitation intensity and by special technical adaptations photodamage could be avoided successfully. For functional biological imaging, multiphoton excitation provides an excellent tool such as the release of caged compounds in a diffraction-limited volume combined with multiphoton or confocal imaging.

Confocal imaging in living brain slices allows the resolution of submicrometre structures of nerve cells, glia and brain vessels. Imaging living brain slices is in many respects different from conventional fixed histological preparations for which confocal microscopes were designed originally. Several problems (i.e. mechanical and thermal drift, and autofluorescence) resulting from the optical and structural properties of brain slices are discussed. Fluorescent indicators may be used to monitor numerous intracellular parameters such as pH and Ca2+ concentration, but not all of them are equally suitable for this type of work. Genetically engineered fluorescent proteins can be used to visualise the fine dendritic structure of neurones or track particular intracellular structures and proteins. They have also been used to generate indicators for Ca2+, cAMP and other molecules. While conventional chemical indicators can be either loaded into neurones via patch pipettes or as membrane-permeable esters, protein indicators can be expressed in various types of cells using adenoviral vectors. Adenoviral transgenesis can be performed in vitro in both acute slices and organotypic slice cultures. Organotypic slice cultures give excellent optical access to neurones loaded with either conventional fluorescent indicators or transfected with adenovirus to express fluorescent proteins. They are most suitable for experiments where both conventional and genetically engineered indicators are combined. Single photon imaging in brain slices is limited to the superficial layers (∼≤50 mm), while multiphoton excitation has a much greater depth of penetration. However, the overall optical resolution achievable in single photon mode is at least as good as when using multiphoton excitation.

Dendritic spines are morphologically and functionally heterogeneous. Here, we use two-photon imaging of layer V pyramidal neurons in slices from mouse visual cortex to characterize differences in spine calcium dynamics between individual spines. By measuring action potential-evoked [Ca2+]i transients in spines, we find different calcium dynamics in spines from proximal apical and distal apical dendrites. Using a mathematical multi-compartmental model, we demonstrate that these differences are even more pronounced in the absence of exogenous calcium buffers. We also find that these different calcium dynamics cause different susceptibility to synaptic depression in proximal and distal apical synapses, and that modifying calcium decay kinetics in spines changes the expression of long-term depression. We conclude that the location of the spine determines its time window of calcium compartmentalization and degree of calcium-dependent synaptic plasticity. Our results highlight the precision of the design of neocortical neurons.

The strength of synaptic connections in the brain is not fixed, but can be modulated by numerous mechanisms. Traditionally, electrophysiology has been used to characterize connections between neurons. Electrophysiology typically reports the activity of populations of synapses, while most mechanisms of plasticity are thought to operate at the level of single synapses. Recently, two-photon laser scanning microscopy has enabled us to perform optical quantal analysis of individual synapses in intact brain tissue. Here we introduce the basic principle of the two-photon microscope and discuss its main differences compared to the confocal microscope. Using calcium imaging in dendritic spines as an example, we explain the advantages of simultaneous dual-dye imaging for quantitative calcium measurements and address two common problems, dye saturation and background fluorescence subtraction.

In both medical research and diagnostics characterization of biological tissue on the cellular level relies on high-resolution optical microscopy. In most cases, however, tissue is excised for microscopic investigation, in part because conventional microscopes are bulky instruments. Imaging of cells in the intact living organism has been difficult. Over the last decade several groups have developed miniature confocal microscopes that use fibre optics to deliver light to the specimen and to measure either reflected or excited fluorescence light. In addition, two-photon excitation recently has been employed in a small portable ‘fibrescope’. A potential clinical application of these microscope probes is their endoscopic use for optical biopsy of inner organs or guidance of conventional biopsy. As a mobile research tool they may permit imaging of neuronal activity in the brain of awake, behaving animals. Here, we review technological approaches to build miniaturized fluorescence microscopes and discuss their potential applications.

Understanding the relationship of the structure of organs to their function is a key component of integrative physiological research. The structure of the organs of the body is not constant but changes, both during growth and development and under conditions of sustained stress (e.g. high altitude exposure and disease). Recently, powerful new techniques have become available in molecular biology, which promise to provide novel insights into the mechanisms and consequences of these altered structure–function relationships. Conventionally structure–function relationships are studied by microscopic examination of tissue sections. However, drawing conclusions about the three-dimensional structure of an organ based on this two-dimensional information frequently leads to serious errors. The techniques of stereology allow precise and accurate quantification of structural features within three-dimensional organs that relate in a meaningful way to integrated function. For example, knowledge of changes in the total surface area of the capillary endothelium in an organ can be related directly to changes in fluid filtration and permeability, or knowledge of total vessel length and mean radius allows deductions about vascular resistance. Confocal microscopy adds enormously to the power of stereological approaches. It reduces the difficulties and labour involved in obtaining suitable images. Moreover, when used in conjunction with new analytical software, it allows convenient application of stereology to small samples and those in which it is essential to maintain a specific orientation for interpretation. The information obtained will allow us to examine in a quantitative manner the altered structure–function relationships produced by manipulation of single genes and regulatory pathways in whole organisms.