BACKGROUND
camp is an ubiquitous second messenger which regulates a wide variety of cellular events and processes ranging from metabolism and gene expression, cell division and migration, exocytosis and secretion, to memory formation and cardiac contractility  <cit> .
on the one hand, camp-signalling cascades are associated with many vital functions in the cns that can be exemplified by camp-related changes in neuronal activity  <cit> , dendritic and axonal growth  <cit> , spine function  <cit> , synaptic activity and neuronal plasticity  <cit> , and gene expression  <cit>  etc.
on the other hand, inadequate camp signalling may lead to long-term disturbances of neuronal functions  <cit> .
camp-dependent enzymes are well characterized in terms of their affinity, kinetics and mechanisms of their modulation  <cit> .
in order to understand how camp-dependent signalling mechanisms operate in living neurons, appropriate tools are needed to monitor spatial and time-dependent changes of camp levels.
direct imaging of camp became possible only recently.
two classes of probes have been developed – one based on protein kinase a  and utilizing fluorescence resonance energy transfer  between fluorescein and rhodamine  <cit>  or cfp and yfp  <cit> , and the other exploiting the exchange protein directly activated by camp, namely epac  <cit> .
pka-based camp probes have slow response times, making them not very suitable for monitoring the rapid changes in i which occur in living cells.
originally designed epac has good sensitivity and temporal resolution  <cit> .
both features are conserved in epac-1-camps  <cit> , its smaller size allowing it to be incorporated into viral vectors with limited genome sizes such as aav.
in comparison with the common methods of transfection in primary cultures  <cit>  and organotypic slices  <cit> , the efficacy of viral gene transfer in postmitotic neurons is much higher.
we recently used adeno-associated viruses  driving transgene expression by the strictly neuron-specific synapsin  <dig> gene promoter  <cit>  to selectively express gfp in neurons in organotypic brainstem slices  <cit>  containing pre-bötzinger complex , the latter constituting the rhythm-generating part of the respiratory network  <cit> .
an important step in further understanding spatial and functional organisation of the respiratory network now is the expression of fluorescent sensors in respiratory neurons and measuring their responses to physiological stimuli.
respiratory activity is crucially dependent on camp signalling, which can be dynamically modulated in specific physiological and pathological conditions  <cit> .
in this study we aimed to specifically target epac1-camps to brainstem neurons.
we characterised the sensitivity and specificity of the sensor to report intraneuronal camp.
we measured  changes after modulation of adenylate cyclase , phosphodiesterase  and activation of g-protein-coupled metabotropic receptors.
changes were tightly related to calcium transients, which are important for respiratory rhythmogenesis  <cit> .
both depolarisation-triggered ca2+ entry and agonist-induced ca2+ release from endoplasmic reticulum  led to camp increase caused by enhancement of ac activity by calcium.
paired calcium increases produced a non-linear summation of subsequent i increases.
this was abolished after pka inhibition, thus indicating an important role for phosphorylation in the dynamic interplay between ca2+ and camp, previously documented in non-excitable cells  <cit> .
RESULTS
application of the aav-epac1-camps vector resulted in expression of the sensor in many neurons per living brainstem slice .
these neurons were arranged into groups resembling the nuclei characteristic for this section of the prebötc-containing brainstem  <cit> .
neuronal somata displayed brighter fluorescence but neuronal processes were also distinctly resolvable .
these acquisitions sharply contrast to images obtained using conventional fluorescent calcium probes such as fluo- <dig> where only the somata of neurons were clearly visible  <cit> .
the ability of epac1-camps to register i changes was first examined in tests where activities of adenylate cyclase  and phosphodiesterase  were modulated.
applications of  <dig> μm forskolin ,  <dig> μm isobutylmethylxanthine ,  <dig> μm rolipram , and  <dig>  mm 2'5'-dideoxyadenosine  induced i changes in line with the presumed drug targets .
effects induced by the drugs were reversible and the responses could be repeatedly elicited after washing-out for  <dig> to  <dig> min.
rolipram was the most effective among pde inhibitors – the increases in i induced by pde <dig> inhibitor milrinone  and pde <dig> inhibitor ehna  were  <dig>  ±  <dig>  and  <dig>  ±  <dig>  μm, respectively.
these effects were smaller than those induced by rolipram  and their magnitude correlates well with the efficacy of the drugs in cardiomyocytes  <cit> .
the greater potency of rolipram thus can explain its stimulating effect on the respiratory motor output in vivo  <cit> .
as exemplified by the two bottom panels in fig.
<dig>  activation of g-protein-coupled receptors produced delayed camp increases, showing the actions of serotonin  and the mglur1/ <dig> agonist - <dig> -dihydroxyphenylglycine .
these responses are consistent with the activation of metabotropic serotonin and glutamate receptors, respectively.
forskolin
mean data ± s. e. m. were obtained in  <dig> to  <dig> neurons in four different preparations and are presented as changes from the resting camp level .
group i metabotropic glutamate receptors  were activated with - <dig> -dihydroxyphenylglycine .
camp-signalling pathways are often activated by increases of calcium levels  <cit> .
in order to examine crosstalk between calcium and camp, we loaded the aav transduced neurons with fura- <dig>  <cit>  and evoked calcium changes by applying brief membrane depolarisations with high-k+ or by inducing calcium release from internal stores.
the first paradigm is similar to the tetanic stimulation often used in the analysis of neuronal plasticity  <cit> .
fig.
3a illustrates how i started to increase at the peak of the calcium transient and reached its maximum during recovery of calcium levels to the resting value.
calcium release from er induced by activation of metabotropic p2y receptors with  <dig> mm atp  <cit>  also led to delayed i increases.
their amplitude and duration were similar to those induced by membrane depolarisation .
various isoforms of ac and pde are known to be differentially activated by calcium  <cit> .
ca2+-dependent stimulation of pde would decrease i, therefore the effects observed indicate a dominating role of ac modulation in ca2+-dependent increases.
after blockade of ac activity with dda, the amplitude of i transients became smaller , indicating that a decrease in production of camp can promote dephosphorylation of ca2+ channels leading to a decrease in their activity  <cit> .
conversely, calcium transients were significantly increased after a blockade of pde  that causes stimulation of pka and subsequent phosphorylation of the channels .
high-k+
atp
mean data ± s. e. m. were obtained in  <dig> to  <dig> neurons in four different preparations.
thapsigargin and ca2+-free solution were applied for  <dig> min before the treatments.
removal of extracellular ca2+ or introduction of  <dig>  mm cd2+, which blocks all pathways for ca2+ entry into the cell, abolished depolarisation-induced i changes .
the blockade of ca2+ entry did not modify the effects of atp .
this indicates that only calcium release after activation of metabotropic p2y receptors is important and ionotropic p2x receptors are not involved in atp-induced i increases.
the effects were occluded after depletion of er with thapsigargin .
this treatment also reduced the effects caused by membrane depolarisations by about 1/ <dig>  indicating contribution of ca2+-induced ca2+ release  to i responses.
it was recently found in presynaptic boutons of drosophila neurons  <cit>  that ca2+-related epac1-camps responses can be mediated by changes in i. this apparently contradicts the fact that the affinity of the sensor to cgmp  is by about one order of magnitude lower than that for camp  <cit> .
however, the presynaptic calcium transients may have considerably higher amplitude and cause ac inactivation  which would make i changes dominant.
in order to examine whether cgmp changes can underlie, in part, the responses of epac1-camps, we applied guanylyl cyclase inhibitor 1h- <cit> -oxadiazolo- -quinoxalin-1-one , the nos inhibitor n-monomethyl-l-arginine , and the no donor s-nitroso-n-acetylpenicillamine .
these drugs were applied at concentrations at which they showed distinct effects on the respiratory motor output in vivo  <cit> , but they did not modify the signals of epac1-camps .
we observed a mutual interference between ca2+ and camp signalling pathways.
application of paired stimuli produced bigger iresponses during the second stimulation which increased calcium .
such enhancement of the response can be mediated by ca2+-driven stimulation of pka increasing the activity of voltage-sensitive ca2+channels  <cit> .
in order to test this assumption, we applied pka inhibitor h- <dig> ethyl]-5-isoquinoline sulfonamide hydrochloride,  <dig> μm for  <dig> min).
after pretreatment with h- <dig>  the potentiation of secondary i and iincreases was abolished .
the enhancement of i responses was also observed after calcium release from er and corresponding effects were inhibited by h- <dig> .
discussion
many vitally important processes which determine the life and fate of neurons are controlled by camp and ca2+, which often act in parallel  <cit> .
in non-excitable cells the fluctuations of i and i are often interrelated  <cit>  and linked via adenylate cyclase  <cit>  or phosphodiesterase  <cit> .
ac has eight different isoforms which can be stimulated and inhibited by ca2+.
the stimulation is characteristic for ac <dig> and ac <dig> which are abundant in the brain and activated by calmodulin.
all ac isoforms are inhibited by supramicromolar concentrations of ca2+  binding to a low-affinity site in the catalytic ac domain  <cit> .
we recorded calcium transients of much smaller amplitude, therefore a ca2+-mediated inhibition of ac should not play any significant role in the effects reported here.
it may however become significant in domains which are located close to the exit of ca2+ from the channels producing calcium gradients which can reach values as high as  <dig> μm  <cit>  and locally limit the production of camp.
the levels of ca2+ and camp are often changed by various neurotransmitters and i changes may be further differentially decoded downstream through, for example, pka, epac, camp-binding protein  and rap to initiate events such as gene expression and cell differentiation.
pka-mediated phosphorylation of channels is important for normal functioning of the channels that mediate entry of ca2+ into the cytoplasm and its release from internal stores.
these phosphorylation/dephosphorylation reactions thus close a regulatory ca2+/camp feedback.
in comparison to ca2+, we currently know much less about the changes in intracellular camp which can occur in relation to neuronal activity.
increasing applications of camp sensors developed in the last decade  <cit>  to different neuronal preparations will definitely bring new knowledge in this area.
although until now such studies have been made only in some cell lines  <cit>  and isolated neurons in primary culture  <cit> , they have already revealed the complexity of camp dynamics.
the neurons in living tissue present the next level of sophistication, as their properties can be modified by interactions with neighbouring neurons and surrounding glial cells.
intrinsic electrical activity of neurons add another important factor, because ensuing calcium fluxes of different amplitude and time-courses and spontaneous calcium release from internal stores should substantially modulate spatial i patterns on different time scales.
how they are involved in different vital cellular functions such as neuronal plasticity, differentiation and development remains to be established.
currently available camp sensors are bulky proteins and the first obstacle in camp imaging concerns the delivery of the probe into the cytoplasm.
a straightforward way is a single cell injection  <cit>  with well known difficulties.
transgenic animals would be an ideal solution and this approach has been successfully used to study camp signalling in the heart  <cit> , pancreatic islets  <cit>  and the neurons of fruit-flies  <cit> .
non-cytotoxic viral gene transfer presents an alternative not limited in its application by the animal or tissue  <cit> .
it can be applied to any cell population given that the sensor is targeted to the specific cell type.
we used a strictly neuron-specific promoter  <cit>  to deliver the camp sensor  <cit>  into neurons in slices.
as a proof of concept, we showed that epac1-camps is expressed in many neurons and reports i levels and their fluctuations in μm range after various physiological stimuli.
we observed that membrane depolarisation and calcium release from internal stores produced i increases that led to further enhancement of calcium entry or calcium release through pka-dependent phosphorylation.
the effects were observed after relatively long-lasting calcium increases similar to those evoked by the tetanic stimulation commonly used to induce a long-term potentiation  <cit> .
the concerted actions of the two second messengers can convey specific messages between neurons but ca2+ and camp should have different spatial and temporal ranges of actions.
for example, the production and consumption rates of camp as well as its diffusion coefficient  are more consistent with slower and spatially extended changes, whereas the actions of ca2+ must be faster and more localised.
how the effects of these two second messengers are orchestrated, and how they influence the neuronal activity, represents a challenging task for future studies.
we believe that neuronal specificity, optical stability and sensitivity of epac1-camps-based sensor provide a solid platform for such examinations.
CONCLUSIONS
we here report the application of epac1-camps to measure camp concentrations in mammalian neurons embedded in their three dimensional context in living tissue.
the sensitivity and specificity of neuron-targeted epac1-camps are characterized and the probe was applied to examine crosstalk between calcium and camp signalling in brainstem neurons.

 camp is an ubiquitous second messenger which regulates a wide variety of cellular events and processes ranging from metabolism and gene expression, cell division and migration, exocytosis and secretion, to memory formation and cardiac contractility. on the one hand, camp-signalling cascades are associated with many vital functions in the cns that can be exemplified by camp-related changes in neuronal activity, dendritic and axonal growth, spine function, synaptic activity and neuronal plasticity, and gene expression etc. on the other hand, inadequate camp signalling may lead to long-term disturbances of neuronal functions. camp-dependent enzymes are well characterized in terms of their affinity, kinetics and mechanisms of their modulation. in order to understand how camp-dependent signalling mechanisms operate in living neurons, appropriate tools are needed to monitor spatial and time-dependent changes of camp levels. direct imaging of camp became possible only recently. two classes of probes have been developed – one based on protein kinase a and utilizing fluorescence resonance energy transfer between fluorescein and rhodamine or cfp and yfp, and the other exploiting the exchange protein directly activated by camp, namely epac. pka-based camp probes have slow response times, making them not very suitable for monitoring the rapid changes in i which occur in living cells. originally designed epac has good sensitivity and temporal resolution. both features are conserved in epac-1-camps, its smaller size allowing it to be incorporated into viral vectors with limited genome sizes such as aav. in comparison with the common methods of transfection in primary cultures and organotypic slices, the efficacy of viral gene transfer in postmitotic neurons is much higher. we recently used adeno-associated viruses driving transgene expression by the strictly neuron-specific synapsin gene promoter to selectively express gfp in neurons in organotypic brainstem slices containing pre-bötzinger complex, the latter constituting the rhythm-generating part of the respiratory network. an important step in further understanding spatial and functional organisation of the respiratory network now is the expression of fluorescent sensors in respiratory neurons and measuring their responses to physiological stimuli. respiratory activity is crucially dependent on camp signalling, which can be dynamically modulated in specific physiological and pathological conditions. in this study we aimed to specifically target epac1-camps to brainstem neurons. we characterised the sensitivity and specificity of the sensor to report intraneuronal camp. we measured changes after modulation of adenylate cyclase, phosphodiesterase and activation of g-protein-coupled metabotropic receptors. changes were tightly related to calcium transients, which are important for respiratory rhythmogenesis. both depolarisation-triggered ca2+ entry and agonist-induced ca2+ release from endoplasmic reticulum led to camp increase caused by enhancement of ac activity by calcium. paired calcium increases produced a non-linear summation of subsequent i increases. this was abolished after pka inhibition, thus indicating an important role for phosphorylation in the dynamic interplay between ca2+ and camp, previously documented in non-excitable cells.  application of the aav-epac1-camps vector resulted in expression of the sensor in many neurons per living brainstem slice. these neurons were arranged into groups resembling the nuclei characteristic for this section of the prebötc-containing brainstem. neuronal somata displayed brighter fluorescence but neuronal processes were also distinctly resolvable. these acquisitions sharply contrast to images obtained using conventional fluorescent calcium probes such as fluo- where only the somata of neurons were clearly visible. the ability of epac1-camps to register i changes was first examined in tests where activities of adenylate cyclase and phosphodiesterase were modulated. applications of μm forskolin, μm isobutylmethylxanthine, μm rolipram, and mm 2'5'-dideoxyadenosine induced i changes in line with the presumed drug targets. effects induced by the drugs were reversible and the responses could be repeatedly elicited after washing-out for to min. rolipram was the most effective among pde inhibitors – the increases in i induced by pde inhibitor milrinone and pde inhibitor ehna were ± and ± μm, respectively. these effects were smaller than those induced by rolipram and their magnitude correlates well with the efficacy of the drugs in cardiomyocytes. the greater potency of rolipram thus can explain its stimulating effect on the respiratory motor output in vivo. as exemplified by the two bottom panels in fig. activation of g-protein-coupled receptors produced delayed camp increases, showing the actions of serotonin and the mglur1/ agonist - -dihydroxyphenylglycine. these responses are consistent with the activation of metabotropic serotonin and glutamate receptors, respectively. forskolin mean data ± s. e. m. were obtained in to neurons in four different preparations and are presented as changes from the resting camp level. group i metabotropic glutamate receptors were activated with - -dihydroxyphenylglycine. camp-signalling pathways are often activated by increases of calcium levels. in order to examine crosstalk between calcium and camp, we loaded the aav transduced neurons with fura- and evoked calcium changes by applying brief membrane depolarisations with high-k+ or by inducing calcium release from internal stores. the first paradigm is similar to the tetanic stimulation often used in the analysis of neuronal plasticity. fig. 3a illustrates how i started to increase at the peak of the calcium transient and reached its maximum during recovery of calcium levels to the resting value. calcium release from er induced by activation of metabotropic p2y receptors with mm atp also led to delayed i increases. their amplitude and duration were similar to those induced by membrane depolarisation. various isoforms of ac and pde are known to be differentially activated by calcium. ca2+-dependent stimulation of pde would decrease i, therefore the effects observed indicate a dominating role of ac modulation in ca2+-dependent increases. after blockade of ac activity with dda, the amplitude of i transients became smaller, indicating that a decrease in production of camp can promote dephosphorylation of ca2+ channels leading to a decrease in their activity. conversely, calcium transients were significantly increased after a blockade of pde that causes stimulation of pka and subsequent phosphorylation of the channels. high-k+ atp mean data ± s. e. m. were obtained in to neurons in four different preparations. thapsigargin and ca2+-free solution were applied for min before the treatments. removal of extracellular ca2+ or introduction of mm cd2+, which blocks all pathways for ca2+ entry into the cell, abolished depolarisation-induced i changes. the blockade of ca2+ entry did not modify the effects of atp. this indicates that only calcium release after activation of metabotropic p2y receptors is important and ionotropic p2x receptors are not involved in atp-induced i increases. the effects were occluded after depletion of er with thapsigargin. this treatment also reduced the effects caused by membrane depolarisations by about 1/ indicating contribution of ca2+-induced ca2+ release to i responses. it was recently found in presynaptic boutons of drosophila neurons that ca2+-related epac1-camps responses can be mediated by changes in i. this apparently contradicts the fact that the affinity of the sensor to cgmp is by about one order of magnitude lower than that for camp. however, the presynaptic calcium transients may have considerably higher amplitude and cause ac inactivation which would make i changes dominant. in order to examine whether cgmp changes can underlie, in part, the responses of epac1-camps, we applied guanylyl cyclase inhibitor 1h- -oxadiazolo- -quinoxalin-1-one, the nos inhibitor n-monomethyl-l-arginine, and the no donor s-nitroso-n-acetylpenicillamine. these drugs were applied at concentrations at which they showed distinct effects on the respiratory motor output in vivo, but they did not modify the signals of epac1-camps. we observed a mutual interference between ca2+ and camp signalling pathways. application of paired stimuli produced bigger iresponses during the second stimulation which increased calcium. such enhancement of the response can be mediated by ca2+-driven stimulation of pka increasing the activity of voltage-sensitive ca2+channels. in order to test this assumption, we applied pka inhibitor h- ethyl]-5-isoquinoline sulfonamide hydrochloride, μm for min). after pretreatment with h- the potentiation of secondary i and iincreases was abolished. the enhancement of i responses was also observed after calcium release from er and corresponding effects were inhibited by h-. discussion many vitally important processes which determine the life and fate of neurons are controlled by camp and ca2+, which often act in parallel. in non-excitable cells the fluctuations of i and i are often interrelated and linked via adenylate cyclase or phosphodiesterase. ac has eight different isoforms which can be stimulated and inhibited by ca2+. the stimulation is characteristic for ac and ac which are abundant in the brain and activated by calmodulin. all ac isoforms are inhibited by supramicromolar concentrations of ca2+ binding to a low-affinity site in the catalytic ac domain. we recorded calcium transients of much smaller amplitude, therefore a ca2+-mediated inhibition of ac should not play any significant role in the effects reported here. it may however become significant in domains which are located close to the exit of ca2+ from the channels producing calcium gradients which can reach values as high as μm and locally limit the production of camp. the levels of ca2+ and camp are often changed by various neurotransmitters and i changes may be further differentially decoded downstream through, for example, pka, epac, camp-binding protein and rap to initiate events such as gene expression and cell differentiation. pka-mediated phosphorylation of channels is important for normal functioning of the channels that mediate entry of ca2+ into the cytoplasm and its release from internal stores. these phosphorylation/dephosphorylation reactions thus close a regulatory ca2+/camp feedback. in comparison to ca2+, we currently know much less about the changes in intracellular camp which can occur in relation to neuronal activity. increasing applications of camp sensors developed in the last decade to different neuronal preparations will definitely bring new knowledge in this area. although until now such studies have been made only in some cell lines and isolated neurons in primary culture, they have already revealed the complexity of camp dynamics. the neurons in living tissue present the next level of sophistication, as their properties can be modified by interactions with neighbouring neurons and surrounding glial cells. intrinsic electrical activity of neurons add another important factor, because ensuing calcium fluxes of different amplitude and time-courses and spontaneous calcium release from internal stores should substantially modulate spatial i patterns on different time scales. how they are involved in different vital cellular functions such as neuronal plasticity, differentiation and development remains to be established. currently available camp sensors are bulky proteins and the first obstacle in camp imaging concerns the delivery of the probe into the cytoplasm. a straightforward way is a single cell injection with well known difficulties. transgenic animals would be an ideal solution and this approach has been successfully used to study camp signalling in the heart, pancreatic islets and the neurons of fruit-flies. non-cytotoxic viral gene transfer presents an alternative not limited in its application by the animal or tissue. it can be applied to any cell population given that the sensor is targeted to the specific cell type. we used a strictly neuron-specific promoter to deliver the camp sensor into neurons in slices. as a proof of concept, we showed that epac1-camps is expressed in many neurons and reports i levels and their fluctuations in μm range after various physiological stimuli. we observed that membrane depolarisation and calcium release from internal stores produced i increases that led to further enhancement of calcium entry or calcium release through pka-dependent phosphorylation. the effects were observed after relatively long-lasting calcium increases similar to those evoked by the tetanic stimulation commonly used to induce a long-term potentiation. the concerted actions of the two second messengers can convey specific messages between neurons but ca2+ and camp should have different spatial and temporal ranges of actions. for example, the production and consumption rates of camp as well as its diffusion coefficient are more consistent with slower and spatially extended changes, whereas the actions of ca2+ must be faster and more localised. how the effects of these two second messengers are orchestrated, and how they influence the neuronal activity, represents a challenging task for future studies. we believe that neuronal specificity, optical stability and sensitivity of epac1-camps-based sensor provide a solid platform for such examinations.  we here report the application of epac1-camps to measure camp concentrations in mammalian neurons embedded in their three dimensional context in living tissue. the sensitivity and specificity of neuron-targeted epac1-camps are characterized and the probe was applied to examine crosstalk between calcium and camp signalling in brainstem neurons. 
these effects were abolished after inhibition of protein kinase a with h- pointing to the important role of phosphorylation of calcium channels in the potentiation of i transients. we recorded i changes in the micromolar range after modulation of adenylate cyclase, inhibition of phosphodiesterase and activation of g-protein-coupled metabotropic receptors. using viral gene transfer we showed its efficient expression in organotypic brainstem preparations. ca2+-dependent i transients were suppressed after blockade of adenylate cyclase with mm adenylate cyclase inhibitor 2'5'-dideoxyadenosine and potentiated after inhibiting phosphodiesterase with isobutylmethylxanthine and rolipram. i levels increased after membrane depolarisation and release of ca2+ from internal stores. using a strictly neuron-restricted promoter we virally transduced neurons in the organotypic brainstem slices which contained pre-bötzinger complex, constituting the rhythm-generating part of the respiratory network. during paired stimulations, the second depolarisation and ca2+ release evoked bigger camp responses. fluorescent camp sensor epac1-camps was expressed both in neuronal cell bodies and neurites, allowing us to measure intracellular distribution of camp, its absolute levels and time-dependent changes in response to physiological stimuli. while imaging of calcium is commonly used in neuroscience applications, probing for camp levels has not yet been performed in living vertebrate neuronal tissue before.
 camp is an ubiquitous second messenger mediating various neuronal functions, often as a consequence of increased intracellular ca2+ levels. while imaging of calcium is commonly used in neuroscience applications, probing for camp levels has not yet been performed in living vertebrate neuronal tissue before.  using a strictly neuron-restricted promoter we virally transduced neurons in the organotypic brainstem slices which contained pre-bötzinger complex, constituting the rhythm-generating part of the respiratory network. fluorescent camp sensor epac1-camps was expressed both in neuronal cell bodies and neurites, allowing us to measure intracellular distribution of camp, its absolute levels and time-dependent changes in response to physiological stimuli. we recorded i changes in the micromolar range after modulation of adenylate cyclase, inhibition of phosphodiesterase and activation of g-protein-coupled metabotropic receptors. i levels increased after membrane depolarisation and release of ca2+ from internal stores. the effects developed slowly and reached their maximum after transient i elevations subsided. ca2+-dependent i transients were suppressed after blockade of adenylate cyclase with mm adenylate cyclase inhibitor 2'5'-dideoxyadenosine and potentiated after inhibiting phosphodiesterase with isobutylmethylxanthine and rolipram. during paired stimulations, the second depolarisation and ca2+ release evoked bigger camp responses. these effects were abolished after inhibition of protein kinase a with h- pointing to the important role of phosphorylation of calcium channels in the potentiation of i transients.  we constructed and characterized a neuron-specific camp probe based on epac1-camps. using viral gene transfer we showed its efficient expression in organotypic brainstem preparations. strong fluorescence, resistance to photobleaching and possibility of direct estimation of levels using dual wavelength measurements make the probe useful in studies of neurons and the mechanisms of their plasticity. epac1-camps was applied to examine the crosstalk between ca2+ and camp signalling and revealed a synergism of actions of these two second messengers. 