Colocalization of protein kinase A with adenylyl cyclase enhances protein kinase A activity during induction of long-lasting long-term-potentiation

The ability of neurons to differentially respond to specific temporal and spatial input patterns underlies information storage in neural circuits. One means of achieving spatial specificity is to restrict signaling molecules to particular subcellular compartments using anchoring molecules such as A-Kinase Anchoring Proteins (AKAPs). Disruption of protein kinase A (PKA) anchoring to AKAPs impairs a PKA-dependent form of long term potentiation (LTP) in the hippocampus. To investigate the role of localized PKA signaling in LTP, we developed a stochastic reaction-diffusion model of the signaling pathways leading to PKA activation in CA1 pyramidal neurons. Simulations investigated whether the role of anchoring is to locate kinases near molecules that activate them, or near their target molecules. The results show that anchoring PKA with adenylyl cyclase (which produces cAMP that activates PKA) produces significantly greater PKA activity, and phosphorylation of both inhibitor-1 and AMPA receptor GluR1 subunit on S845, than when PKA is anchored apart from adenylyl cyclase. The spatial microdomain of cAMP was smaller than that of PKA suggesting that anchoring PKA near its source of cAMP is critical because inactivation by phosphodiesterase limits diffusion of cAMP. The prediction that the role of anchoring is to colocalize PKA near adenylyl cyclase was confirmed by experimentally rescuing the deficit in LTP produced by disruption of PKA anchoring using phosphodiesterase inhibitors. Additional experiments confirm the model prediction that disruption of anchoring impairs S845 phosphorylation produced by forskolin-induced synaptic potentiation. Collectively, these results show that locating PKA near adenylyl cyclase is a critical function of anchoring.     

Multiple neural oscillators and muscle feedback are required for the intestinal fed state motor program

After a meal, the gastrointestinal tract exhibits a set of behaviours known as the fed state. A major feature of the fed state is a little understood motor pattern known as segmentation, which is essential for digestion and nutrient absorption. Segmentation manifests as rhythmic local constrictions that do not propagate along the intestine. In guinea-pig jejunum in vitro segmentation constrictions occur in short bursts together with other motor patterns in episodes of activity lasting 40-60 s and separated by quiescent episodes lasting 40-200 s. This activity is induced by luminal nutrients and abolished by blocking activity in the enteric nervous system (ENS). We investigated the enteric circuits that regulate segmentation focusing on a central feature of the ENS: a recurrent excitatory network of intrinsic sensory neurons (ISNs) which are characterized by prolonged after-hyperpolarizing potentials (AHPs) following their action potentials. We first examined the effects of depressing AHPs with blockers of the underlying channels (TRAM-34 and clotrimazole) on motor patterns induced in guinea-pig jejunum, in vitro, by luminal decanoic acid. Contractile episode durations increased markedly, but the frequency and number of constrictions within segmenting bursts and quiescent period durations were unaffected. We used these observations to develop a computational model of activity in ISNs, excitatory and inhibitory motor neurons and the muscle. The model predicted that: i) feedback to ISNs from contractions in the circular muscle is required to produce alternating activity and quiescence with the right durations; ii) transmission from ISNs to excitatory motor neurons is via fast excitatory synaptic potentials (EPSPs) and to inhibitory motor neurons via slow EPSPs. We conclude that two rhythm generators regulate segmentation: one drives contractions within segmentation bursts, the other the occurrence of bursts. The latter depends on AHPs in ISNs and feedback to these neurons from contraction of the circular muscle.     

Modulation of the feeding system by a radular mechanosensory neuron in the terrestrial slug,Incilaria fruhstorferi

We investigated the modulatory role of a radular mechanoreceptor (RM) in the feeding system of Incilaria. RM spiking induced by current injection evoked several cycles of rhythmic buccal motor activity in quiescent preparations, and this effect was also observed in preparations lacking the cerebral ganglia. The evoked rhythmic activity included sequential activation of the inframedian radular tensor, the supramedian radular tensor, and the buccal sphincter muscles in that order.In addition to the generation of rhythmic motor activity, RM spiking enhanced tonic activities in buccal nerve 1 as well as in the cerebrobuccal connective, showing a wide excitatory effect on buccal neurons. The excitatory effect was further examined in the supramedian radular tensor motoneuron. RM spiking evoked biphasic depolarization in the tensor motoneuron consisting of fast excitatory postsynaptic potentials and prolonged depolarization lasting after termination of RM spiking. These depolarizations also occurred in high divalent cation saline, suggesting that they were both monosynaptic.When RM spiking was evoked in the fictive rasp phase during food-induced buccal motor rhythm, the activity of the supramedian radular tensor muscle showed the greatest enhancement of the three muscles tested, while the rate of ongoing rhythmic motor activity showed no increase.Abbreviations CPG central pattern generator - EPSP excitatory postsynaptic potential - RBMA rhythmic buccal motor activity - RM radular mechanosensory neuron - SMT supramedian radular tensor neuron     

Synaptic interactions mediating synchrony and oscillations in primate sensorimotor cortex.

The appearance of oscillatory modes of 'gamma' activity in many cortical areas of different species has generated interest in understanding their underlying mechanisms and possible functions. This paper reviews evidence from studies on primate motor cortex showing that oscillatory activity entrains many neurons during periods of exploratory manipulative behavior. These oscillatory episodes synchronize widely spread neurons in sensorimotor cortex bilaterally, including descending corticospinal neurons, as evidenced by correlated modulations in EMG activity. The resulting neural synchronization involves task-related and -unrelated neurons similarly, suggesting that it is more likely to play some global role in attention than mediating any obvious interactions involved in coordinating movements. Intracellular recordings have elucidated the strength and types of synaptic interactions between motor cortical neurons that are involved in both normal and oscillatory activity. Spike-triggered averages (STAs) of intracellular membrane potentials have revealed serial connections in the form of unitary excitatory and inhibitory post-synaptic potentials (EPSPs and IPSPs). More commonly, STAs showed large synchronous excitatory or inhibitory potentials (ASEPs and ASIPs) beginning before the trigger spike and composed of multiple unitary events. ASEPs involved synchronous activity in a larger and more widespread group of presynaptic neurons than ASIPs. During oscillatory episodes synchronized excitatory and inhibitory synaptic potentials occurred in varying proportions. EPSPs evoked by stimulating neighboring cortical sites during the depolarizing phase of spontaneous oscillations showed evidence of transient potentiation. These observations are consistent with several functional hypotheses, but fit best with a possible role in attention or arousal.     

Chemosensory stimulation of the oral cavity of the snail Helisoma

Food extracts, perfused through the oral cavity of the snailHelisoma trivolvis, lead to synaptic activation of identifiedbuccal ganglia motor neurons. Both retractor and protractormotor neurons displayed cyclic bursts of firing characteristicof that observed during expression of the central feeding motorprogram(CFM). The possibility that leakage of food extracts from theoral cavity had a pharmacological effect on buccal neurons wasconsidered. Direct application of the extracts to the exposedganglionic surface did not evoke similar neuronal activity.Oral perfusion with a behaviorally aversive compound inhibitedboth the activity evoked by acceptable taste solutions and "spontaneously"generated activity in some preparations. It is concluded thatoral chemosensory receptors in the snail exert both an excitatoryand inhibitory influence on buccal motor neurons. The significanceof these results for cellular neurophysiological investigationof the synaptic events underlying the central processing ofafferent chemosensory information is discussed.     

Neural mechanism generating firing patterns in jaw motoneurons during the food-induced response in Aplysia kurodai

1. In each right and left buccal ganglia of Aplysia kurodai, we identified 4 premotor neurons impinging on the ipsilateral jaw-closing and -opening motoneurons. Three of them (MA1 neurons) had features of multifunctional neurons. Current-induced spikes in the MA1 neurons produced excitatory junction potentials (EJPs) in the buccal muscle fibers. In addition, tactile stimulation of the buccal muscle surface produced a train of spikes in the MA1 neurons without synaptic input. The other neuron (MA2) had only a premotor function. 2. The MA1 and MA2 neurons had similar synaptic effects on the jaw-closing and -opening motoneurons. Current-induced spikes in the premotor neurons gave rise to monosynaptic inhibitory postsynaptic potentials (IPSPs) in the ipsilateral jaw-closing motoneurons. Simultaneously, spikes in one of the MA1 neurons and the MA2 also gave rise to monosynaptic excitatory postsynaptic potentials (EPSPs) in the ipsilateral jaw-opening motoneuron. 3. The IPSPs and the EPSPs induced by spikes in the premotor neurons were reversibly blocked by d-tubocurarine and hexamethonium, respectively, suggesting that the MA1 and MA2 neurons are cholinergic. 4. When depolarizing and hyperpolarizing current pulses were passed into one premotor neuron, attenuated but similar potential changes were produced in another randomly selected premotor neuron in the same ganglion, suggesting that they are electronically coupled.     

Physiological basis of feeding behavior in Tritonia diomedea. III. Role of depolarizing afterpotentials

The nature and role of the depolarizing afterpotentials (DAPs) of buccal motoneurons of Tritonia diomedea were examined. Neuron B5 exhibits a DAP whose ionic dependence and modifiability by TEA and 4-AP suggest a similarity to the DAP previously described in pleural pacemaker neurons. Reduction of the DAP severely reduces the ability of these neurons to generate bursts of action potentials. Certain other motoneurons (B1 and B6) are reexcited by a slow DAP (SDAP) which appears to be of synaptic origin. It is concluded that DAPs, which are dependent upon motoneuron activity, contribute to the synthesis of motor output by the buccal ganglion.     

Amiloride inhibits contraction and serotonin modulation of Aplysia muscle

1. Serotonin (5-HT) potentiates acetylcholine (ACh)-elicited contractions of Aplysia buccal muscles. Serotonin potentiation was significantly reduced by 0.03 mM, 0.1 mM, and 0.3 mM amiloride. 2. Unpotentiated ACh-elicited contractions were significantly reduced by 0.1 mM and 0.3 mM amiloride. 3. Amiloride reduced ACh-elicited depolarization. The reduction in contraction caused by 0.3 mM amiloride (to 16% of control) was larger than could be explained by the reduction in depolarization (86% of control). 4. Amiloride had no effect on tension in skinned muscle fibers, indicating that amiloride probably did not have a direct effect on contractile mechanisms. 5. Potentiation of contraction produced by zero sodium (Tris substituted, 0 Na-Tris) medium could be abolished by 0.3 mM amiloride. 6. Zero Na-Tris increased 45Ca influx 2.7-fold. In the presence of 0.3 mM amiloride, 0 Na-Tris increased 45Ca influx only 1.4-fold. 7. Amiloride (0.3 mM) reduced the elevation of muscle cAMP caused by 10(-6) M 5-HT by 60%. Zero Na-Tris did not cause a change in muscle cAMP.     

Ingestion motor programs of Aplysia are modulated by short-term synaptic enhancement in cerebral-buccal interneuron pathways

In the sea slug Aplysia, buccal synapses of cerebral-buccal interneurons (CBIs) CBI-2 and CBI-12 exhibit short-term synaptic enhancement (STE), including frequency-dependant facilitation and augmentation/post-tetanic potentiation (AUG/PTP). The STE that results from driving CBI-2 or CBI-12 is associated with significantly decreased latency to burst onset in buccal premotor neurons and motor neurons, increased cycle frequency of ingestion buccal motor programs (iBMPs) and increased intraburst firing frequency of buccal neurons during iBMPs. Tests of paired-pulse facilitation during AUG/PTP suggest that the locus for this plasticity is presynaptic. The AUG/PTP is not elicited by heterosynaptic pathways, indicating that its origin is homosynaptic. At low CBI-2 and CBI-12 firing frequencies, STE is likely to contribute to iBMP initiation, while at higher firing frequencies, STE is correlated with increased cycle frequency of iBMPs. Thus, STE is an important component of the mechanisms whereby cerebral neurons regulate cyclic feeding programs and likely contributes to observed variations in behavioral responses, including feeding arousal. Electronic Publication     

Neuromuscular adjustments that constrain submaximal EMG amplitude at task failure of sustained isometric contractions

The amplitude of the surface EMG does not reach the level achieved during a maximal voluntary contraction force at the end of a sustained, submaximal contraction, despite near-maximal levels of voluntary effort. The depression of EMG amplitude may be explained by several neural and muscular adjustments during fatiguing contractions, including decreased net neural drive to the muscle, changes in the shape of the motor unit action potentials, and EMG amplitude cancellation. The changes in these parameters for the entire motor unit pool, however, cannot be measured experimentally. The present study used a computational model to simulate the adjustments during sustained isometric contractions and thereby determine the relative importance of these factors in explaining the submaximal levels of EMG amplitude at task failure. The simulation results indicated that the amount of amplitude cancellation in the simulated EMG (～ 40%) exhibited a negligible change during the fatiguing contractions. Instead, the main determinant of the submaximal EMG amplitude at task failure was a decrease in muscle activation (number of muscle fiber action potentials), due to a reduction in the net synaptic input to motor neurons, with a lesser contribution from changes in the shape of the motor unit action potentials. Despite the association between the submaximal EMG amplitude and reduced muscle activation, the deficit in EMG amplitude at task failure was not consistently associated with the decrease in neural drive (number of motor unit action potentials) to the muscle. This indicates that the EMG amplitude cannot be used as an index of neural drive.     

Influence of timing variability between motor unit potentials on M-wave characteristics

The transient enlargement of the compound muscle action potential (M wave) after a conditioning contraction is referred to as potentiation. It has been recently shown that the potentiation of the first and second phases of a monopolar M wave differed drastically; namely, the first phase remained largely unchanged, whereas the second phase underwent a marked enlargement and shortening. This dissimilar potentiation of the first and second phases has been suggested to be attributed to a transient increase in conduction velocity after the contraction. Here, we present a series of simulations to test if changes in the timing variability between motor unit potentials (MUPs) can be responsible for the unequal potentiation (and shortening) of the first and the second M-wave phases. We found that an increase in the mean motor unit conduction velocity resulted in a marked enlargement and narrowing of both the first and second M-wave phases. The enlargement of the first phase caused by a global increase in motor unit conduction velocities was apparent even for the electrode located over the innervation zone and became more pronounced with increasing distance to the innervation zone, whereas the potentiation of the second phase was largely independent of electrode position. Our simulations indicate that it is unlikely that an increase in motor unit conduction velocities (accompanied or not by changes in their distribution) could account for the experimental observation that only the second phase of a monopolar M wave, but not the first, is enlarged after a brief contraction. However, the combination of an increase in the motor unit conduction velocities and a spreading of the motor unit activation times could potentially explain the asymmetric potentiation of the M-wave phases.     

Trans-spinal direct current enhances corticospinal output and stimulation-evoked release of glutamate analog, D-2,3-³H-aspartic acid

Trans-spinal direct current (tsDC) stimulation is a modulator of spinal excitability and can influence cortically elicited muscle contraction in a polarity-dependent fashion. When combined with low-frequency repetitive cortical stimulation, cathodal tsDC [tsDC(-)] produces a long-term facilitation of cortically elicited muscle actions. We investigated the ability of this combined stimulation paradigm to facilitate cortically elicited muscle actions in spinal cord-injured and noninjured animals. The effect of tsDC-applied alone or in combination with repetitive spinal stimulation (rSS) on the release of the glutamate analog, D-2,3-(3)H-aspartate (D-Asp), from spinal cord preparations in vitro-was also tested. In noninjured animals, tsDC (-2 mA) reproducibly potentiated cortically elicited contractions of contralateral and ipsilateral muscles tested at various levels of baseline muscle contraction forces. Cortically elicited muscle responses in animals with contusive and hemisectioned spinal cord injuries (SCIs) were similarly potentiated. The combined paradigm of stimulation caused long-lasting potentiation of cortically elicited bilateral muscle contraction in injured and noninjured animals. Additional analysis suggests that at higher baseline forces, tsDC(-) application does not increase the rising slope of the muscle contraction but causes repeated firing of the same motor units. Both cathodal and anodal stimulations induced a significant increase of D-Asp release in vitro. The effect of the combined paradigm of stimulation (tsDC and rSS) on the concentration of extracellular D-Asp was polarity dependent. These results indicate that tsDC can powerfully modulate the responsiveness of spinal cord neurons. The results obtained from the in vitro preparation suggest that the changes in neuronal excitability were correlated with an increased concentration of extracellular glutamate. The combined paradigm of stimulation, used in our experiments, could be noninvasively applied to restore motor control in humans with SCI.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Activity of fusimotor neurons during reflex muscle contraction

The influence of fusimotor activity via the gamma-loop on reflex responses of motoneurons to stretch or vibration stimulation of mm. triceps surae was studied in decerebrate cats. Action potentials of single fusimotor neurons were derived from thin filaments isolated from nerves innervating this muscle group, leaving their main nerve supply intact. Most fusimotor neurons tested were found to be coactivated with motor units during reflex muscle contraction. In the initial period of development of reflex muscle contraction a weak autogenetic inhibitory effect on discharge of fusimotor neurons was found. The results suggest that reduction of the reflex motor signal, leading to a "silent period," is partly the result of a transient decrease in the fusimotor output effect on contracting muscles. A study of changes in fusimotor discharge generation during the ascending phase of reflex muscle contraction may provide data useful for identification of autogenetic reflex influences on these motoneurons and for elucidating the conditions necessary for servoassistance of muscle contractions.Medical Research Institute, Belgrade, Yugoslavia. Translated from Neirofiziologiya, Vol. 16, No. 5, pp. 630–637, September–October, 1984.     

Ultrastructure and electrical properties of the hyperneural muscle of Periplaneta americana

The hyperneural muscle of Periplaneta americana is striated with an A-band at least 2 μm long. Z-bands were discrete units, but arranged with some order in the myoplasm. The sarcoplasmic reticulum was reduced and 1 thick was surrounded by 10 to 12 thin myofilaments. The muscle is innervated from the median nerves by axons containing electron-dense granules which may be opaque near the neuromuscular junction amid numerous synaptic vesicles. Depolarizing intracellular current injection produces an ohmic voltage response of the membrane potential and neurally evoked contraction is effected by summated excitatory postsynaptic potentials. All contractal activity ceases when the innervation is removed. The muscle appears to be electrically inexcitable and to be under obligatory control of central motor units. By virtue of attachments along the ventral nerve cord in the abdomen, the hyperneural muscle, when activated, moves the nerve cord unidirectionally. In effect, the hyperneural muscle is called upon by the central nervous system to move the ventral nerve cord presumably into a greater mix with the haemolymph in response to as yet unknown stimuli.     

K(+)-channel blockers restore synaptic plasticity in the neuromuscular junction of dunce, a Drosophila learning and memory mutant.

The effects of K(+)-channel blockers on synaptic transmission in dunce (dnc), a Drosophila learning and memory mutant, were investigated. Larvae dnc mutants lack facilitation and post-tetanic potentiation (PTP) at their motor end-plates; dnc mutants are also deficient in a form of phosphodiesterase, and exhibit abnormally high levels of cyclic adenosine 3',5'-monophosphate (cAMP). A two-microelectrode voltage-clamp was used to record end-plate currents and spontaneous end-plate currents from longitudinal ventrolateral third-instar larval muscle. The K(+)-channel blockers 3,4-diaminopyridine (3,4-DAP) and tetraethylammonium (TEA), at micromolar concentrations, caused a reversible decrease in end-plate current amplitudes both in wild-type and mutant end-plates. In the presence of blockers, a period of high-frequency stimulation (tetanus) of the nerve gave way to a transient increase in the end-plate currents of dnc mutants resembling facilitation and PTP in normal end-plates; 3,4-DAP and TEA also restored facilitation and PTP in normal end-plates after incubation with a non-hydrolysable analogue of cAMP (8Br-cAMP). It is suggested that a specific K+ conductance might be relevant to the lack of synaptic plasticity at the dnc neuromuscular synapses.     

Coping with variability in small neuronal networks

Experimental and corresponding modeling studies indicate that there is a 2- to 5-fold variation of intrinsic and synaptic parameters across animals while functional output is maintained. Here, we review experiments, using the heartbeat central pattern generator (CPG) in medicinal leeches, which explore the consequences of animal-to-animal variation in synaptic strength for coordinated motor output. We focus on a set of segmental heart motor neurons that all receive inhibitory synaptic input from the same four premotor interneurons. These four premotor inputs fire in a phase progression and the motor neurons also fire in a phase progression because of differences in synaptic strength profiles of the four inputs among segments. Our work tested the hypothesis that functional output is maintained in the face of animal-to-animal variation in the absolute strength of connections because relative strengths of the four inputs onto particular motor neurons is maintained across animals. Our experiments showed that relative strength is not strictly maintained across animals even as functional output is maintained, and animal-to-animal variations in strength of particular inputs do not correlate strongly with output phase. Further experiments measured the precise temporal pattern of the premotor inputs, the segmental synaptic strength profiles of their connections onto motor neurons, and the temporal pattern (phase progression) of those motor neurons all in the same animal for a series of 12 animals. The analysis of input and output in this sample of 12 individuals suggests that the number (four) of inputs to each motor neuron and the variability of the temporal pattern of input from the CPG across individuals weaken the influence of the strength of individual inputs. Moreover, the temporal pattern of the output varies as much across individuals as that of the input. Essentially, each animal arrives at a unique solution for how the network produces functional output.