cAMP signaling affects age-associated deterioration of pacemaker beating interval dynamics.

Sinoatrial node (SAN) beating interval variability (BIV) and the average beating interval (BI) are regulated by a coupled-clock system, driven by Ca2+-calmodulin activated adenylyl cyclase, cAMP, and downstream PKA signaling. Reduced responsiveness of the BI and BIV to submaximal, [X]50, ß-adrenergic receptor (ß-AR) stimulation, and phosphodiesterase inhibition (PDEI) have been documented in aged SAN tissue, whereas the maximal responses, [X]max, do not differ by age. To determine whether age-associated dysfunction in cAMP signaling leads to altered responsiveness of BI and BIV, we measured cAMP levels and BI in adult (2-4 months n = 27) and aged (22-26 months n = 25) C57/BL6 mouse SAN tissue in control and in response to ß-AR or PDEI at X50 and [X]max. Both cAMP and average BI in adult SAN were reduced at X50, whereas cAMP and BI at Xmax did not differ by age. cAMP levels and average BI were correlated both within and between adult and aged SAN. BIV parameters in long- and short-range terms were correlated with cAMP levels for adult SAN. However, due to reduced cAMP within aged tissues at [X]50, these correlations were diminished in advanced age. Thus, cAMP level generated by the coupled clock mechanisms is tightly linked to average BI. Reduced cAMP level at X50 in aged SAN explains the reduced responsiveness of the BI and BIV to ß-AR stimulation and PDEI.

Changes in cAMP signaling are associated with age-related downregulation of spontaneously beating atrial tissue energetic indices.

The prevalence of atria-related diseases increases exponentially with age and is associated with ATP supply-to-demand imbalances. Because evidence suggests that cAMP regulates ATP supply-to-demand, we explored aged-associated alterations in atrial ATP supply-to-demand balance and its correlation with cAMP levels. Right atrial tissues driven by spontaneous sinoatrial node impulses were isolated from aged (22-26 months) and adult (3-4 months) C57/BL6 mice. ATP demand increased by addition of isoproterenol or 3-Isobutyl-1-methylxanthine (IBMX) and decreased by application of carbachol. Each drug was administrated at the dose that led to a maximal change in beating rate (Xmax) and to 50% of that maximal change in adult tissue (X50). cAMP, NADH, NAD + NADH, and ATP levels were measured in the same tissue. The tight correlation between cAMP levels and the beating rate (i.e., the ATP demand) demonstrated in adult atria was altered in aged atria. cAMP levels were lower in aged compared to adult atrial tissue exposed to X50 of ISO or IBMX, but this difference narrowed at Xmax. Neither ATP nor NADH levels correlated with ATP demand in either adult or aged atria. Baseline NADH levels were lower in aged as compared to adult atria, but were restored by drug perturbations that increased cAMP levels. Reduction in Ca2+-activated adenylyl cyclase-induced decreased cAMP and prolongation of the spontaneous beat interval of adult atrial tissue to their baseline levels in aged tissue, brought energetics indices to baseline levels in aged tissue. Thus, cAMP regulates right atrial ATP supply-to-demand matching and can restore age-associated ATP supply-to-demand imbalance.

cAMP-PKA signaling modulates the automaticity of human iPSC-derived cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been used to screen and characterize drugs and to reveal mechanisms underlying cardiac diseases. However, before hiPSC-CMs can be used as a reliable experimental model, the physiological mechanisms underlying their normal function should be further explored. Accordingly, a major feature of hiPSC-CMs is automaticity, which is regulated by both Ca2+ and membrane clocks. To investigate the mechanisms coupling these clocks, we tested three hypotheses: (1) normal automaticity of spontaneously beating hiPSC-CMs is regulated by local Ca2+ releases (LCRs) and cAMP/PKA-dependent coupling of Ca2+ clock to M clock; (2) the LCR period indicates the level of crosstalk within the coupled-clock system; and (3) perturbing the activity of even one clock can lead to hiPSC-CM-altered automaticity due to diminished crosstalk within the coupled-clock system. By measuring the local and global Ca2+ transients, we found that the LCRs properties are correlated with the spontaneous beat interval. Changes in cAMP-dependent coupling of the Ca2+ and M clocks, caused by a pharmacological intervention that either activates the ß-adrenergic or cholinergic receptor or upregulates/downregulates PKA signaling, affected LCR properties, which in turn altered hiPSC-CMs automaticity. Clocks' uncoupling by attenuating the pacemaker current If or the sarcoplasmic reticulum Ca2+ kinetics, decreased hiPSC-CMs beating rate, and prolonged the LCR period. Finally, LCR characteristics of spontaneously beating (at comparable rates) hiPSC-CMs and rabbit SAN are similar. In conclusion, hiPSC-CM automaticity is controlled by the coupled-clock system whose function is mediated by Ca2+-cAMP-PKA signaling.

Induced neuro-vascular interactions robustly enhance functional attributes of engineered neural implants.

Engineered neural implants have a myriad of potential basic science and clinical neural repair applications. Although there are implants that are currently undergoing their first clinical investigations, optimizing their long-term viability and efficacy remain an open challenge. Functional implants with pre-vascularization of various engineered tissues have proven to enhance post-implantation host integration, and well-known synergistic neural-vascular interplays suggest that this strategy could also be promising for neural tissue engineering. Here, we report the development of a novel bio-engineered neuro-vascular co-culture construct, and demonstrate that it exhibits enhanced neurotrophic factor expression, and more complex neuronal morphology. Crucially, by introducing genetically encoded calcium indicators (GECIs) into the co-culture, we are able to monitor functional activity of the neural network, and demonstrate greater activity levels and complexity as a result of the introduction of endothelial cells in the construct. The presence of this enhanced activity could putatively lead to superior integration outcomes. Indeed, leveraging on the ability to monitor the construct's development post-implantation with GECIs, we observe improved integration phenotypes in the spinal cord of mice relative to non-vascularized controls. Our approach provides a new experimental system with functional neural feedback for studying the interplay between vascular and neural development while advancing the optimization of neural implants towards potential clinical applications.

All-optical bidirectional neural interfacing using hybrid multiphoton holographic optogenetic stimulation.

Our understanding of neural information processing could potentially be advanced by combining flexible three-dimensional (3-D) neuroimaging and stimulation. Recent developments in optogenetics suggest that neurophotonic approaches are in principle highly suited for noncontact stimulation of network activity patterns. In particular, two-photon holographic optical neural stimulation (2P-HONS) has emerged as a leading approach for multisite 3-D excitation, and combining it with temporal focusing (TF) further enables axially confined yet spatially extended light patterns. Here, we study key steps toward bidirectional cell-targeted 3-D interfacing by introducing and testing a hybrid new 2P-TF-HONS stimulation path for accurate parallel optogenetic excitation into a recently developed hybrid multiphoton 3-D imaging system. The system is shown to allow targeted all-optical probing of in vitro cortical networks expressing channelrhodopsin-2 using a regeneratively amplified femtosecond laser source tuned to 905 nm. These developments further advance a prospective new tool for studying and achieving distributed control over 3-D neuronal circuits both in vitro and in vivo.

Holographic fiber bundle system for patterned optogenetic activation of large-scale neuronal networks.

Optogenetic perturbation has become a fundamental tool in controlling activity in neurons. Used to control activity in cell cultures, slice preparations, anesthetized and awake behaving animals, optical control of cell-type specific activity enables the interrogation of complex systems. A remaining challenge in developing optical control tools is the ability to produce defined light patterns such that power-efficient, precise control of neuronal populations is obtained. Here, we describe a system for patterned stimulation that enables the generation of structured activity in neurons by transmitting optical patterns from computer-generated holograms through an optical fiber bundle. The system couples the optical system to versatile fiber bundle configurations, including coherent or incoherent bundles composed of hundreds of up to several meters long fibers. We describe the components of the system, a method for calibration, and a detailed power efficiency and spatial specificity quantification. Next, we use the system to precisely control single-cell activity as measured by extracellular electrophysiological recordings in ChR2-expressing cortical cell cultures. The described system complements recent descriptions of optical control systems, presenting a system suitable for high-resolution spatiotemporal optical control of wide-area neural networks in vitro and in vivo, yielding a tool for precise neural system interrogation.

Hybrid multiphoton volumetric functional imaging of large-scale bioengineered neuronal networks.

Planar neural networks and interfaces serve as versatile in vitro models of central nervous system physiology, but adaptations of related methods to three dimensions (3D) have met with limited success. Here, we demonstrate for the first time volumetric functional imaging in a bioengineered neural tissue growing in a transparent hydrogel with cortical cellular and synaptic densities, by introducing complementary new developments in nonlinear microscopy and neural tissue engineering. Our system uses a novel hybrid multiphoton microscope design combining a 3D scanning-line temporal-focusing subsystem and a conventional laser-scanning multiphoton microscope to provide functional and structural volumetric imaging capabilities: dense microscopic 3D sampling at tens of volumes per second of structures with mm-scale dimensions containing a network of over 1,000 developing cells with complex spontaneous activity patterns. These developments open new opportunities for large-scale neuronal interfacing and for applications of 3D engineered networks ranging from basic neuroscience to the screening of neuroactive substances.

Holographically patterned activation using photo-absorber induced neural-thermal stimulation.

OBJECTIVE: Patterned photo-stimulation offers a promising path towards the effective control of distributed neuronal circuits. Here, we demonstrate the feasibility and governing principles of spatiotemporally patterned microscopic photo-absorber induced neural-thermal stimulation (PAINTS) based on light absorption by exogenous extracellular photo-absorbers. APPROACH: We projected holographic light patterns from a green continuous-wave (CW) or an IR femtosecond laser onto exogenous photo-absorbing particles dispersed in the vicinity of cultured rat cortical cells. Experimental results are compared to predictions of a temperature-rate model (where membrane currents follow I ∝ dT/dt). MAIN RESULTS: The induced microscopic photo-thermal transients have sub-millisecond thermal relaxation times and stimulate adjacent cells. PAINTS activation thresholds for different laser pulse durations (0.02 to 1 ms) follow the Lapicque strength-duration formula, but with different chronaxies and minimal threshold energy levels for the two excitation lasers (an order of magnitude lower for the IR system <50 nJ). Moreover, the empirical thresholds for the CW system are found to be in good agreement with detailed simulations of the temperature-rate model, but are generally lower for the IR system, suggesting an auxiliary excitation mechanism. SIGNIFICANCE: Holographically patterned PAINTS could potentially provide a means for minimally intrusive control over neuronal dynamics with a high level of spatial and temporal selectivity.

Holographic optogenetic stimulation of patterned neuronal activity for vision restoration.

When natural photoreception is disrupted, as in outer-retinal degenerative diseases, artificial stimulation of surviving nerve cells offers a potential strategy for bypassing compromised neural circuits. Recently, light-sensitive proteins that photosensitize quiescent neurons have generated unprecedented opportunities for optogenetic neuronal control, inspiring early development of optical retinal prostheses. Selectively exciting large neural populations are essential for eliciting meaningful perceptions in the brain. Here we provide the first demonstration of holographic photo-stimulation strategies for bionic vision restoration. In blind retinas, we demonstrate reliable holographically patterned optogenetic stimulation of retinal ganglion cells with millisecond temporal precision and cellular resolution. Holographic excitation strategies could enable flexible control over distributed neuronal circuits, potentially paving the way towards high-acuity vision restoration devices and additional medical and scientific neuro-photonics applications.

Learning-induced enhancement of feedback inhibitory synaptic transmission.

Olfactory-discrimination learning results with a series of intrinsic and excitatory synaptic modifications in piriform cortex pyramidal neurons. Here we show that such learning results with long-lasting enhancement of inhibitory synaptic transmission onto proximal dendrites of these pyramidal neurons. Such enhancement is mediated by a strong hyperpolarizing shift in the reversal potential of fast inhibitory postsynaptic potentials (fIPSPs). Moreover, paired-pulse depression of these IPSPs, indicating enhanced GABA release, is also apparent after learning. We suggest that learning is accompanied by long-lasting enhancement of synaptic inhibition onto excitatory neurons, thus compensating for the increase of excitation in these neurons.

Learning-induced modulation of SK channels-mediated effect on synaptic transmission.

Although small conductance (SK)-mediated calcium-dependent potassium currents are usually mostly thought to modulate neuronal adaptation by suppressing repetitive spike firing, recent evidence suggests that these channels also modulate synaptic transmission. SK2 channels were shown to be activated in dendritic spines following calcium entry via N-methyl-d-aspartate (NMDA) receptor. Such activation of potassium currents terminates the NMDA-dependent postsynaptic potential (PSP). Synaptic potentials in pyramidal neurons in the piriform cortex from olfactory-discrimination-trained rats have enhanced rise time 3 days after learning, and their dendritic spines are significantly smaller at this time. In the present study we examined whether the SK channel-mediated effect on PSPs is modified after learning. The SK channels inhibitor, apamin, that selectively blocks the SK channels-mediated potassium currents enhanced the width of the PSP in neurons from trained rats only. This effect is abolished in the presence of the NMDA-channel blocker, APV. The learning-induced reduction in paired-pulse facilitation was not affected by apamin. Although the effect of the SK channels is increased after learning, the protein expression level of the SK2 channels, the type located in dendritic spines, was decreased after learning. The protein expression level of the SK3 channel, suggested to be located mainly in axon terminals, was not modified by learning. We suggest that the enhanced effect of the SK channels on NMDA-mediated synaptic transmission is the result of the reduction in the spine volume after learning. Moreover, these data indicate that spines are more excitable after learning, and are thus more predisposed to activity-dependent modifications.

A novel role for extracellular signal-regulated kinase in maintaining long-term memory-relevant excitability changes.

Pyramidal neurons in the piriform cortex from olfactory-discrimination-trained rats show enhanced intrinsic neuronal excitability that lasts for several days after learning. Such enhanced intrinsic excitability is mediated by long-term reduction in the postburst afterhyperpolarization (AHP), which is generated by repetitive spike firing. AHP reduction is attributable to decreased conductance of a calcium-dependent potassium current, the sI(AHP). We have previously shown that such learning-induced AHP reduction is maintained by PKC activation. However, the molecular machinery underlying such long-lasting modulation of intrinsic excitability is yet to be fully described. Here we examine whether the extracellular signal-regulated kinase I/II (ERKI/II) pathway, which is known to be crucial in learning, memory, and synaptic plasticity processes, is instrumental for the long-term maintenance of learning-induced AHP reduction. PD98059 or UO126, which selectively block MEK, the upstream kinase of ERK, increased the AHP in neurons from trained rats but not in neurons from naive and pseudo-trained rats. Consequently, the differences in AHP amplitude and neuronal adaptation between neurons from trained rats and controls were abolished. This effect was not mediated by modulation of basic membrane properties. In accordance with its effect on neuronal excitability, the level of activated ERK in the membranal fraction was significantly higher in piriform cortex samples taken from trained rats. In addition, the PKC activator OAG (1-oleoyl-20acety-sn-glycerol), which was shown to reduce the AHP in neurons from control rats, had no effect on these neurons in the presence of PD98059. Our data show that ERK has a key role in maintaining long-lasting learning-induced enhancement of neuronal excitability.

Learning-induced reversal of the effect of noradrenalin on the postburst AHP.

Pyramidal neurons in the piriform cortex from olfactory-discrimination-trained rats have reduced postburst afterhyperpolarization (AHP), for 3 days after learning, and are thus more excitable during this period. Such AHP reduction is caused by decreased conductance of one or more of the calcium-dependent potassium currents, I(AHP) and sI(AHP), that mediate the medium and slow AHPs. In this study, we examined which potassium current is reduced by learning and how the effect of noradrenalin (NE) on neuronal excitability is modified by such reduction. The small conductance (SK) channels inhibitor, apamin, that selectively blocks I(A)(HP), reduced the AHP in neurons from trained, naïve, and pseudotrained rats to a similar extent, thus maintaining the difference in AHP amplitude between neurons from trained rats and controls. In addition, the protein expression level of the SK1, SK2, and SK3 channels was also similar in all groups. NE, which was shown to enhance I(AHP) while suppressing (S)I(AHP), reduced the AHP in neurons from controls but enhanced the AHP in neurons from trained rats. Our data show that learning-induced enhancement of neuronal excitability is not the result of reduction in the I(AHP) current. Thus it is probably mediated by reduction in conductance of the other calcium-dependent potassium current, sI(AHP). Consequently, the effect of NE on neuronal excitability is reversed. We propose that the change in the effect of NE after learning may act to counterbalance learning-induced hyperexcitability and preserve the piriform cortex ability to subserve olfactory learning.

A molecular mechanism for stabilization of learning-induced synaptic modifications.

Olfaction is a principal sensory modality in rodents, and rats quickly learn to discriminate between odors and to associate odor with reward. Here we show that such olfactory discrimination (OD) learning consists of two phases with distinct cellular mechanisms: an initial NMDAR-sensitive phase in which the animals acquire a successful behavioral strategy (rule learning), followed by an NMDAR-insensitive phase in which the animals learn to distinguish between individual odors (pair learning). Rule learning regulates the composition of synaptic NMDARs in the piriform cortex, resulting in receptors with a higher complement of the NR2a subunit protein relative to NR2b. Rule learning also reduces long-term potentiation (LTP) induced by high-frequency stimulation of the intracortical axons in slices of piriform cortex. As NR2a-containing NMDARs mediate shorter excitatory postsynaptic currents than those containing NR2b, we suggest that learning-induced regulation of NMDAR composition constrains subsequent synaptic plasticity, thereby maintaining the memory encoded by experience.

Learning-induced long-term synaptic modifications in the olfactory cortex.

The idea that memory is manifested at the cellular level by enhancement of synaptic connections between simultaneously activated neurons has been suggested half a century ago by Hebb, and is widely accepted since. Much effort is done to describe such enhancement and reveal the underlying mechanisms. Learning-induced synaptic modifications were studied in the last decade with in-vitro brain slices preparations. Several forms of long-term enhancement of synaptic connections between layer II pyramidal neurons in the piriform cortex accompany olfactory learning. Such modifications were described also in other brain areas, following other training paradigms. Post-synaptic enhancement of synaptic transmission is indicated by reduced rise time of (post synaptic potentials) PSPs and formation of new synaptic connections is indicated by increased spine density along dendrites of these neurons. Enhanced synaptic release is indicated by reduced paired-pulse facilitation. In slices from trained rats predisposition for long-term potentiation is decreased and predisposition for long-term depression is increased. These modifications are attributed to olfactory-discrimination rule learning, rather than to memories for specific odors, and may be subsequent to intrinsic modifications in pyramidal neurons that create favorable conditions for activity-dependent synaptic enhancement.

Learning-induced reduction in post-burst after-hyperpolarization (AHP) is mediated by activation of PKC.

We studied the role of protein kinase C (PKC) and protein kinase A (PKA) in mediating learning-related long lasting reduction of the post-burst after-hyperpolarization (AHP) in cortical pyramidal neurons. We have shown previously that pyramidal neurons in the rat piriform (olfactory) cortex from trained (TR) rats have reduced post-burst AHP for 3 days after odour-discrimination learning, and that this reduction is due to decreased conductance of calcium-dependent potassium current. In the present study, we examined whether this long-lasting reduction in AHP is mediated by second messenger systems. The broad-spectrum kinase inhibitor, H7, increased the AHP in neurons from TR rats, but not in neurons from pseudo-trained (pseudo-TR) and naive rats. Consequently, the difference in AHP amplitude between neurons from TR and control animals was diminished. This effect was also obtained by application of the specific PKC inhibitor, GF-109203x. The PKC activator, 1-Oleoyl-2-acetyl-sn-glycerol (OAG), significantly reduced the AHP in neurons from naive and pseudo-TR rats, but not in neurons from TR rats, so that the difference between the groups was abolished. The PKA-specific inhibitor, H-89, increased the AHP in neurons from all groups to a similar extent, and the difference in AHP amplitude between neurons from TR rats and neurons from controls was maintained. We suggest that while the post-burst AHP in piriform cortex pyramidal neurons is modulated by both PKC and PKA, a PKC-dependent process maintains the learning-related reduction of the AHP in these cells.