Microchannel electrode interfaces to assess bladder afferent activity

By placing axons into polymeric micro-channels hosting embedded electrodes the extracellular amplitude of action potentials is greatly increased, allowing for robust recording and noise suppression. We are developing such an electrode interface to record electrical activity from bladder afferents to restore bladder control in patients suffering from spinal cord injury. Here we describe our microchannel electrode interface in terms of design, microfabrication and electrode characteristics and report on in vivo bladder function after implantation of teased dorsal rootlets within microchannels.

A microchannel neuroprosthesis for bladder control after spinal cord injury in rat

A severe complication of spinal cord injury is loss of bladder function (neurogenic bladder), which is characterized by loss of bladder sensation and voluntary control of micturition (urination), and spontaneous hyperreflexive voiding against a closed sphincter (detrusor-sphincter dyssynergia). A sacral anterior root stimulator at low frequency can drive volitional bladder voiding, but surgical rhizotomy of the lumbosacral dorsal roots is needed to prevent spontaneous voiding and dyssynergia. However, rhizotomy is irreversible and eliminates sexual function, and the stimulator gives no information on bladder fullness. We designed a closed-loop neuroprosthetic interface that measures bladder fullness and prevents spontaneous voiding episodes without the need for dorsal rhizotomy in a rat model. To obtain bladder sensory information, we implanted teased dorsal roots (rootlets) within the rat vertebral column into microchannel electrodes, which provided signal amplification and noise suppression. As long as they were attached to the spinal cord, these rootlets survived for up to 3 months and contained axons and blood vessels. Electrophysiological recordings showed that half of the rootlets propagated action potentials, with firing frequency correlated to bladder fullness. When the bladder became full enough to initiate spontaneous voiding, high-frequency/amplitude sensory activity was detected. Voiding was abolished using a high-frequency depolarizing block to the ventral roots. A ventral root stimulator initiated bladder emptying at low frequency and prevented unwanted contraction at high frequency. These data suggest that sensory information from the dorsal root together with a ventral root stimulator could form the basis for a closed-loop bladder neuroprosthetic. Copyright © 2013, American Association for the Advancement of Science

Proteomics of the neurotoxic fraction from the sea anemone Bunodosoma cangicum venom: Novel peptides belonging to new classes of toxins

In contrast to the many studies on the venoms of scorpions, spiders, snakes and cone snails, tip to now there has been no report of the proteomic analysis of sea anemones venoms. In this work we report for the first time the peptide mass fingerprint and some novel peptides in the neurotoxic fraction (Fr III) of the sea anemone Bunodosoma cangicum venom. Fr III is neurotoxic to crabs and was purified by rp-HPLC in a C-18 column, yielding 41 fractions. By checking their molecular masses by ESI-Q-Tof and MALDI-Tof MS we found 81 components ranging from near 250 amu to approximately 6000 amu. Some of the peptidic molecules were partially sequenced through the automated Edman technique. Three of them are peptides with near 4500 amu belonging to the class of the BcIV, BDS-I, BDS-II, APETx1, APETx2 and Am-II toxins. Another three peptides represent a novel group of toxins (similar to 3200 amu). A further three molecules (similar to similar to 4900 amu) belong to the group of type 1 sodium channel neurotoxins. When assayed over the crab leg nerve compound action potentials, one of the BcIV- and APETx-like peptides exhibits an action similar to the type 1 sodium channel toxins in this preparation, suggesting the same target in this assay. On the other hand one of the novel peptides, with 3176 amu, displayed an action similar to potassium channel blockage in this experiment. In summary, the proteomic analysis and mass fingerprint of fractions from sea anemone venoms through MS are valuable tools, allowing us to rapidly predict the occurrence of different groups of toxins and facilitating the search and characterization of novel molecules without the need of full characterization of individual components by broader assays and bioassay-guided purifications. It also shows that sea anemones employ dozens of components for prey capture and defense. (C) 2008 Elsevier Inc. All rights reserved.

Eugenol modifies the excitability of rat sciatic nerve and superior cervical ganglion neurons

Eugenol is a phenylpropene obtained from the essential oils of plants such as clove and basil which has ample use in dentistry. Eugenol possesses analgesic effects that may be related to the inhibition of voltage-dependent Na(+) channels and/or to the activation of TRPV1 receptors or both. In the present study, electrophysiological parameters were taken from the compound action potentials of the isolated rat sciatic nerve and from neurons of the superior cervical ganglion (SCG) impaled with sharp microelectrodes under current-clamp conditions. In the isolated rat sciatic nerve, eugenol inhibited the compound action potential in a concentration-dependent manner. Action potentials recorded from SCG neurons were inhibited by eugenol with an IC(50) of 0.31 mM. At high concentrations (2 mM), during brief applications. eugenol caused significant action potential blockade while it did not interfere with the resting membrane potential or the membrane input resistance. Surprisingly, however, at low eugenol concentrations (0.6 mM), during long time applications, a reversible reduction (by about 50%) in the input membrane resistance was observed, suggesting the possible involvement of a secondary delayed effect of eugenol to reduce neuronal excitability. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

An investigation of Hebbian phase sequences as assembly graphs

Hebb proposed that synapses between neurons that fire synchronously are strengthened, forming cell assemblies and phase sequences. The former, on a shorter scale, are ensembles of synchronized cells that function transiently as a closed processing system; the latter, on a larger scale, correspond to the sequential activation of cell assemblies able to represent percepts and behaviors. Nowadays, the recording of large neuronal populations allows for the detection of multiple cell assemblies. Within Hebb's theory, the next logical step is the analysis of phase sequences. Here we detected phase sequences as consecutive assembly activation patterns, and then analyzed their graph attributes in relation to behavior. We investigated action potentials recorded from the adult rat hippocampus and neocortex before, during and after novel object exploration (experimental periods). Within assembly graphs, each assembly corresponded to a node, and each edge corresponded to the temporal sequence of consecutive node activations. The sum of all assembly activations was proportional to firing rates, but the activity of individual assemblies was not. Assembly repertoire was stable across experimental periods, suggesting that novel experience does not create new assemblies in the adult rat. Assembly graph attributes, on the other hand, varied significantly across behavioral states and experimental periods, and were separable enough to correctly classify experimental periods (Naïve Bayes classifier; maximum AUROCs ranging from 0.55 to 0.99) and behavioral states (waking, slow wave sleep, and rapid eye movement sleep; maximum AUROCs ranging from 0.64 to 0.98). Our findings agree with Hebb's view that assemblies correspond to primitive building blocks of representation, nearly unchanged in the adult, while phase sequences are labile across behavioral states and change after novel experience. The results are compatible with a role for phase sequences in behavior and cognition.

Abdominal hyperalgesia in secretory phospholipase A(2)-induced rat pancreatitis: Distinct roles of NK1 receptors

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Dinâmica da Plasticidade Sináptica em neurônios do hipocampo durante ciclos de sono: um estudo computacional

Several research lines show that sleep favors memory consolidation and learning. It has been proposed that the cognitive role of sleep is derived from a global scaling of synaptic weights, able to homeostatically restore the ability to learn new things, erasing memories overnight. This phenomenon is typical of slow-wave sleep (SWS) and characterized by non-Hebbian mechanisms, i.e., mechanisms independent of synchronous neuronal activity. Another view holds that sleep also triggers the specific enhancement of synaptic connections, carrying out the embossing of certain mnemonic traces within a lattice of synaptic weights rescaled each night. Such an embossing is understood as the combination of Hebbian and non-Hebbian mechanisms, capable of increasing and decreasing respectively the synaptic weights in complementary circuits, leading to selective memory improvement and a restructuring of synaptic configuration (SC) that can be crucial for the generation of new behaviors ( insights ). The empirical findings indicate that initiation of Hebbian plasticity during sleep occurs in the transition of the SWS to the stage of rapid eye movement (REM), possibly due to the significant differences between the firing rates regimes of the stages and the up-regulation of factors involved in longterm synaptic plasticity. In this study the theories of homeostasis and embossing were compared using an artificial neural network (ANN) fed with action potentials recorded in the hippocampus of rats during the sleep-wake cycle. In the simulation in which the ANN did not apply the long-term plasticity mechanisms during sleep (SWS-transition REM), the synaptic weights distribution was re-scaled inexorably, for its mean value proportional to the input firing rate, erasing the synaptic weights pattern that had been established initially. In contrast, when the long-term plasticity is modeled during the transition SWSREM, an increase of synaptic weights were observed in the range of initial/low values, redistributing effectively the weights in a way to reinforce a subset of synapses over time. The results suggest that a positive regulation coming from the long-term plasticity can completely change the role of sleep: its absence leads to forgetting; its presence leads to a positive mnemonic change

Indicadores de cálcio e de voltagem codificados geneticamente na detecção de potenciais de ação e inputs sinápticos em cultura de neurônios hipocampais

Recently, genetically encoded optical indicators have emerged as noninvasive tools of high spatial and temporal resolution utilized to monitor the activity of individual neurons and specific neuronal populations. The increasing number of new optogenetic indicators, together with the absence of comparisons under identical conditions, has generated difficulty in choosing the most appropriate protein, depending on the experimental design. Therefore, the purpose of our study was to compare three recently developed reporter proteins: the calcium indicators GCaMP3 and R-GECO1, and the voltage indicator VSFP butterfly1.2. These probes were expressed in hippocampal neurons in culture, which were subjected to patchclamp recordings and optical imaging. The three groups (each one expressing a protein) exhibited similar values of membrane potential (in mV, GCaMP3: -56 ±8.0, R-GECO1: -57 ±2.5; VSFP: -60 ±3.9, p = 0.86); however, the group of neurons expressing VSFP showed a lower average of input resistance than the other groups (in Mohms, GCaMP3: 161 ±18.3; GECO1-R: 128 ±15.3; VSFP: 94 ±14.0, p = 0.02). Each neuron was submitted to current injections at different frequencies (10 Hz, 5 Hz, 3 Hz, 1.5 Hz, and 0.7 Hz) and their fluorescence responses were recorded in time. In our study, only 26.7% (4/15) of the neurons expressing VSFP showed detectable fluorescence signal in response to action potentials (APs). The average signal-to-noise ratio (SNR) obtained in response to five spikes (at 10 Hz) was small (1.3 ± 0.21), however the rapid kinetics of the VSFP allowed discrimination of APs as individual peaks, with detection of 53% of the evoked APs. Frequencies below 5 Hz and subthreshold signals were undetectable due to high noise. On the other hand, calcium indicators showed the greatest change in fluorescence following the same protocol (five APs at 10 Hz). Among the GCaMP3 expressing neurons, 80% (8/10) exhibited signal, with an average SNR value of 21 ±6.69 (soma), while for the R-GECO1 neurons, 50% (2/4) of the neurons had signal, with a mean SNR value of 52 ±19.7 (soma). For protocols at 10 Hz, 54% of the evoked APs were detected with GCaMP3 and 85% with R-GECO1. APs were detectable in all the analyzed frequencies and fluorescence signals were detected from subthreshold depolarizations as well. Because GCaMP3 is the most likely to yield fluorescence signal and with high SNR, some experiments were performed only with this probe. We demonstrate that GCaMP3 is effective in detecting synaptic inputs (involving Ca2+ influx), with high spatial and temporal resolution. Differences were also observed between the SNR values resulting from evoked APs, compared to spontaneous APs. In recordings of groups of cells, GCaMP3 showed clear discrimination between activated and silent cells, and reveals itself as a potential tool in studies of neuronal synchronization. Thus, our results indicate that the presently available calcium indicators allow detailed studies on neuronal communication, ranging from individual dendritic spines to the investigation of events of synchrony in neuronal networks genetically defined. In contrast, studies employing VSFPs represent a promising technology for monitoring neural activity and, although still to be improved, they may become more appropriate than calcium indicators, since neurons work on a time scale faster than events of calcium may foresee

MOTOR NERVE CONDUCTION and REPETITIVE NERVE STIMULATION IN CAPTIVE RING-TAILED COATI (NASUA NASUA)

There are few electrophysiologic studies in wild animals. The aim of this study was to determine normal data for motor nerve conduction studies and repetitive stimulation in sciatic-tibial and ulnar nerves in clinically normal captive coati. Eight adult ring-tailed coatis (Nasua nasua), two females and six males weighing 68 kg, were used. Average nerve conduction velocity was 70.81 m/sec (standard deviation [SD] = 3.98) and 56.93 m/sec (SD = 4.31) for the sciatic-tibial and ulnar nerves, respectively. Repetitive stimulation responses demonstrated minimal variations of the area of the compound muscle action potentials at low (3 Hz) and high (20 Hz) frequencies. The maximal obtained decremental area response was 8%. These normal data of conduction studies may be used in assessing abnormalities for clinical diagnosis. In addition, the obtained normal repetitive stimulation data were similar to dogs and humans and may be used for post- and presynaptic disturbances of the neuromuscular transmission in coatis.

Padronização da determinação da velocidade de condução nervosa sensitiva dos nervos tibial e peroneal de cães clinicamente sadios, pela utilização de eletrodos de superfície

O presente trabalho teve como objetivo a padronização dos valores de referência de velocidade de condução nervosa sensitiva dos nervos tibial e peroneal em cães clinicamente sadios, pela utilização de eletrodos de superfície. em todos os sítios de estimulação, captação, referência e terra foram utilizados eletrodos do tipo jacaré, exceto na captação do estímulo no nervo peroneal, próximo à articulação fêmur-tibial, onde o registro só foi possível com a utilização de eletrodo de agulha. Foram utilizados 30 cães, 11 machos e 19 fêmeas, sem raça definida, com idade entre dois e seis anos. Os valores médios das medidas dos potenciais evocados pela estimulação sensitiva dos nervos tibial e peroneal foram: latência inicial, 1,82±0,30ms (1,30 a 2,55ms) e 1,57±0,29ms (1,01 a 2,16ms), amplitude de pico a pico, 96,48±45,78miV (41,6 a 214miV) e 121,25±57,49miV (54,8 a 299miV) e duração, 1,97±0,69ms (1,01 a 3,56ms) e 2,37±0,85ms (1,11 a 3,94ms), respectivamente. Os valores médios das medidas de velocidade de condução nervosa sensitiva dos nervos tibial e peroneal foram, respectivamente, 62,14+7,71ms (50,0 a 77,2ms) e 65,18+6,42ms (53,8 a 79,2ms), respectivamente.

Avaliação eletroneuromiográfica em gatos normais e submetidos ao hiperparatireoidismo secundário nutricional

O trabalho teve por objetivos estudar a condução nervosa motora e a transmissão neuromuscular e eletromiografia de repouso em gatos normais (grupo I), submetidos a hiperparatireoidismo secundário nutricional (grupo II). Para estudo normativo (grupo I), foram utilizados 10 gatos, aparentemente saudáveis, sem raça definida, sendo seis machos e quatro fêmeas, com idades entre 4 e 5 meses e peso médio de 1,67kg. No grupo II, empregaram-se 10 gatos, sem raça definida, sendo cinco machos e cinco fêmeas, com idade aproximada inicial entre 2 e 3 meses e peso inicial médio de 820 gramas. Após um período de adaptação de 10 dias, foram alimentados por 60 dias com coração bovino moído e cru, visando a indução de hiperparatireoidismo secundário nutricional. Foi possível concluir que latência, amplitude e velocidade de condução nervosa motora e os achados eletromiográficos das atividades insercional e espontânea de gatos com hiperparatireoisdismo secundário nutricional, apresentaram um padrão similar aos de gatos normais da mesma idade. Para estimulações repetitivas a 3Hz, observou-se tendência global a decremento dos potenciais de ação musculares compostos e a 10 Hz houve tendência de incremento ou decremento; entretanto, tais variações apresentaram-se dentro dos limites de normalidade.

Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Differential effects of novel wasp toxin on rat hippocampal interneurons

We studied the effects of a wasp toxin beta-pompilidotoxin (beta-PMTX) on rat hippocampal CA1 interneurons by the current-clamp technique. The firing patterns of pyramidal neurons and pyramidale interneurons were not affected by beta-PMTX, but in oriens and radiatum interneurons, beta-PMTX converted the action potentials to prolonged depolarizing potentials by slowing the inactivation of Na+ channels. In lacunosum moleculare interneurons, beta-PMTX induced initial bursting spikes followed by block of succeeding spikes. Comparison of beta-PMTX with a sea anemone toxin, ATX 11, revealed that ATX 11 altered the firing properties of pyramidal neurons and pyramidale interneurons that were unchanged by beta-PMTX. Our results suggest that beta-PMTX modulates Na+ currents in CAl interneurons differently in various CAl neurons and the toxin is useful to classify Na+ channel subtypes. (C) 2002 Elsevier B.V. Ireland Ltd. All rights reserved.

A new class of neurotoxin from wasp venom slows inactivation of sodium current

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action or alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX stowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Reduced Insulin Secretion in Protein Malnourished Mice Is Associated with Multiple Changes in the beta-Cell Stimulus-Secretion Coupling

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)