Ultrastructural clarification of the peripherally located actin network in the myelinated axons / Aclaración ultraestructural de la red de actina localizada periféricamente en los axones mielinizados

SUMMARY: Despite the existence of a large amount of actin in the axons, the concentration F-actin was quite low in the myelinated axons and almost all the F-actin were located in the peripheries of the myelinated axons. Until now, the ultrastructural localization of F-actin has still not been reported in the myelinated axons, probably due to the lack of an appropriate detection method. In the present study, a phalloidin-based FITC-anti-FITC technique was adopted to investigate the subcellular localization of F-actin in the myelinated axons. By using this technique, F-actin is located in the outer and inner collars of myelinated cytoplasm surrounding the intermodal axon, the Schmidt-Lanterman incisures, the paranodal terminal loops and the nodal microvilli. In addition, the satellite cell envelope, which encapsulates the axonal initial segment of the peripheral sensory neuron, was also demonstrated as an F-actin-enriched structure. This study provided a hitherto unreported ultrastructural view of the F-actin in the myelinated axons, which may assist in understanding the unique organization of axonal actin cytoskeleton.

RESUMEN: A pesar de la existencia de una gran cantidad de actina en los axones, la concentración de F-actina era bastante baja en los axones mielinizados y casi la totalidad de F-actina se localizaba en las periferias de los axones mielinizados. A la fecha aún no se ha reportado la localización ultraestructural de F-actina en los axones mielinizados, probablemente debido a la falta de un método de detección apropiado. En el presente estudio, se adoptó una técnica FITC-anti-FITC basada en faloidina para investigar la localización subcelular de F-actina en los axones mielinizados. Mediante el uso de esta técnica, la F-actina se localiza en los collares externo e interno del citoplasma mielinizado que rodea el axón intermodal, a las incisiones de Schmidt-Lanterman,a las asas terminales paranodales y a las microvellosidades nodales. Además, la envoltura de la célula satélite, que encapsula el segmento axonal inicial de la neurona sensorial periférica, también se demostró como una estructura enriquecida con F-actina. Este estudio proporcionó una vista ultraestructural de la F-actina en los axones mielinizados, que puede ayudar a comprender la organización única del citoesqueleto de actina axonal.

Anatomical and ultrastructural sex differences in mean diameter and thickness of myelinated axons in adult rat corpus callosum / Diferencias sexuales anatómicas y ultraestructurales del diámetro medio y grosor de los axones mielinizados en el cuerpo calloso de ratas adultas

Sexual dimorphism exists at all levels of the nervous system. These sex differences could underlie genderrelated differences in behavior and neuropsychological function, as well as the gender differences in the prevalence of various mental disorders such as autism, attention deficit disorders, and schizophrenia. Myelination, on the other hand, is a unique cellular process that can have a dramatic impact on the structure and physiology of an axon and its surrounding tissue. The corpus callosum (CC) is the largest of the brain commissures, which connects the cerebral cortices of the two hemispheres, and provides interhemispheric connectivity for information transfer and processing between cortical regions. Variation in the axonal properties of CC will alter the interhemispheric connectivity. The CC consists of myelinated and unmyelinated axons, glial cells and blood vessels. Several functional studies have reported that the function of CC is associated with its axons density and myelination properties. The sexual dimorphism in the axonal content of the CC has always been controversial; hence, the aim of this study was to analyze the differences in axons' diameter and myelin sheath thickness of the CC between male and female rats. For this purpose, five pairs of adult male and female rats were perfused and the CC were removed and sectioned. Four sections from different subregions of the corpus callosum that represent the genu, anterior body, posterior body, and splenium of the CC were stained and electron microscopic images were captured using stereological guidelines. Later, the axons diameter and myelin sheath thickness for each subregion were calculated and compared between males and females. Our preliminary findings of the present study indicated region specific differences in the myelinated axon thickness and diameter in the CC between male and female rats.

El dimorfismo sexual existe en todos los niveles del sistema nervioso. Estas diferencias de sexo podrían ser la base de las diferencias de comportamiento y función neuropsicológica relacionadas con el sexo, así como las diferencias en la prevalencia de diversos trastornos mentales, como el autismo, los trastornos por déficit de atención y la esquizofrenia. La mielinización, por otro lado, es un proceso celular único que puede tener un impacto dramático en la estructura y fisiología de un axón y su tejido circundante. El cuerpo calloso (CC) es la mayor comisura cerebral, que conecta las cortezas cerebrales de ambos hemisferios, y proporciona la conectividad interhemisférica para la transferencia y el procesamiento de información entre regiones corticales. La variación en las propiedades axonales de CC alterará la conectividad interhemisférica. El CC consiste en axones mielinizados y no mielinizados, células gliales y vasos sanguíneos. Varios estudios funcionales han informado que la función de CC está asociada con la densidad de axones y las propiedades de mielinización. El dimorfismo sexual en el contenido axonal del CC siempre ha sido controvertido; por lo tanto, el objetivo de este estudio fue analizar las diferencias en el diámetro de los axones y el grosor de la vaina de mielina del CC entre ratas macho y hembra. Para este propósito, se perfundieron cinco pares de ratas macho y hembra adultas y se extrajeron y seccionaron las CC. Se tiñeron cuatro secciones de diferentes subregiones del cuerpo calloso que representan el genu, el cuerpo anterior, el cuerpo posterior y el esplenio y se capturaron imágenes de microscopía electrónicas utilizando referencias estereológicas. Posteriormente se calculó el diámetro de los axones y el grosor de la vaina de mielina para cada subregión y se compararon entre machos y hembras. Nuestros hallazgos preliminares del presente estudio indicaron diferencias específicas en el grosor y diámetro del axón mielinizado en el CC entre ratas macho y hembra.

A histomorphometric study of unmyelinated fibers of the fibular nerve in Wistar rats / Histomorfometria das fibras amielínicas do nervo fibular em ratos Wistar

ABSTRACT There are few histomorphometric studies on the unmyelinated fibers of the fibular nerve in rats, and the number of experimental studies using this nerve has been increasing in the last years. Sixty-two percent of the endoneurial area from 10 fibular nerves of adult Wistar rats was scanned by electron microscopy, and digitized. The total number of unmyelinated axons (1.882 ± 271) was significantly lesser, and their axon diameters (0.2 µm to 2.8 µm) significantly higher than that determined in previous studies. The histogram peaked at 1 µm. The differences could be due to the nerve sampled area, the number and the age of the animals evaluated, and the laboratory techniques used. This study brings new and referential data to be used in experimental investigations involving histomorphometric evaluation of the rat fibular nerve.

RESUMO Embora o nervo fibular de ratos venha sendo incluído progressivamente em maior número de estudos experimentais nos últimos anos, há poucos estudos a respeito das suas fibras amielínicas. Os nervos fibulares de 10 ratos Wistar adultos foram avaliados através de microscopia óptica e eletrônica. Varredura sistemática através de microscopia eletrônica de transmissão das áreas fasciculares da porção distal no nervo foi realizada. Em média, 62% da área endoneural foi digitalizada. O número total de axônios amielínicos encontrados (1.882 ± 271) foi significativamente menor e as medidas dos diâmetros axonais (0,2 µm a 2,8 µm) maiores do que o determinado em estudos prévios. O pico do histograma foi constituído por fibras de 1µm. As diferenças podem ser devidas à amostragem de maior área endoneural, ao número e à idade dos animais avaliados, e as técnicas laboratoriais utilizadas. Os dados obtidos podem ser considerados referenciais para o nervo fibular de ratos Wistar adultos.

Análisis morfométrico del proceso de diferenciación in vitro de neuronas del hipocampo / Morphometric analysis of the differentiation process of hippocampal neurons in vitro

Los cultivos neuronales del sistema nervioso central se han venido usando ampliamente para el estudio de los mecanismos que conducen el proceso de diferenciación neuronal, así como también se han empleado como modelos in vitro para evaluar drogas y desarrollar nuevas terapias, de allí la importancia profundizar en la caracterización de dicho proceso. En este estudio, se prepararon cultivos primarios de células del hipocampo para estudiar los tipos celulares desarrollados, el desarrollo de dendritas y axones, la densidad de vesículas sinápticas y el desarrollo de los conos de crecimiento. Mediante inmunofluorescencia usando anticuerpos y marcadores no inmunológicos, se observaron los cambios experimentados por las estructuras de interés durante diferentes estadios temporales (1-21 días). Observamos una mayor proporción de neuronas sobre glias, desarrollo normal de las redes neuronales (conformadas por dendritas y axones), incremento en la longitud de dendritas y el establecimiento de sinapsis. Las vesículas sinápticas también experimentaron un incremento en su densidad a medida que aumentaba el tiempo de cultivo. Finalmente, se estudiaron los cambios morfológicos de los conos de crecimiento observándose que al inicio del cultivo en su mayoría se encontraban cerrados, pero a medida que maduraban las neuronas la proporción de conos de crecimiento abiertos aumentó. Este trabajo representa un avance en la caracterización morfométrica de los cultivos neuronales puesto que recoge de manera simultánea y cuantitativa los principales aspectos que marcan el proceso de diferenciación neuronal. En este estudio, la medición de estas características morfológicas hizo posible establecer parámetros cuantitativos que ayudarán a distinguir las principales etapas de la diferenciación neuronal.

Neuronal cultures of the central nervous system are widely used to study the molecular mechanisms that rule the differentiation process. These cultures have also been used to evaluate drugs and to develop new therapies. From this we can infer the relevance of performing an extended characterization that involves the main aspects driving such process. To carry out such characterization in the present study we prepared primary cultures from hippocampal cells to study cell identity, development of neuronal processes (dendrites and axons), density of synaptic vesicles and development of growth cones. Using immunofluorescence techniques, specific antibodies and non-immunological probes, we studied the changes experienced by the structures under study during different temporal stages (1-21 days). We observed a major proportion of neurons over glia, normal development of neuronal networks (formed by dendrites and axons), increase in the length of dendrites and axons and establishment of synaptic connections. Synaptic vesicles also showed an increase in their densities as long as the time of the culture progressed. Finally, we studied the morphological changes of the growth cones and observed that those were mostly closed at the beginning of the culture period. As neurons matured we observed an increase in the proportion of open growth cones. This work represents an advance in the morphometric characterization of neuronal cultures, since it gathers the main aspects that outline the neuronal differentiation process. In this study, measurement of these morphological features made possible to establish quantitative markers that will allow establishing more precisely the different stages of neuronal differentiation.

Watery and dark axons in Wallerian degeneration of the opossum's optic nerve: different patterns of cytoskeletal breakdown?

In this paper we report a qualitative morphological analysis of Wallerian degeneration in a marsupial. Right optic nerves of opossums Didelphis marsupialis were crushed with a fine forceps and after 24, 48, 72, 96 and 168 hours the animals were anaesthetized and perfused with fixative. The optic nerves were immersed in fixative and processed for routine transmission electron microscopy. Among the early alterations typical of axonal degeneration, we observed nerve fibers with focal degeneration of the axoplasmic cytoskeleton, watery degeneration and dark degeneration, the latter being prevalent at 168 hours after crush. Our results point to a gradual disintegration of the axoplasmic cytoskeleton, opposed to the previous view of an "all-or-nothing'' process (Griffin et al 1995). We also report that, due to an unknown mechanism, fibers show either a dark or watery pattern of axonal degeneration, as observed in axon profiles. We also observed fibers undergoing early myelin breakdown in the absence of axonal alterations

Further observations on cerebellar climbing fibers. A study by means of light microscopy, confocal laser scanning microscopy and scanning and transmission electron microscopy

The intracortical pathways of climbing fibers were traced in several vertebrate cerebella using light microscopy, confocal laser scanning microscopy, scanning and transmission electron microscopy. They were identified as fine fibers up to 1(micron thick, with a characteristic crossing-over bifurcation pattern. Climbing fiber collaterals were tridimensionally visualized forming thin climbing fiber glomeruli in the granular layer. Confocal laser scanning microscopy revealed three types of collateral processes at the interface between granular and Purkinje cell layers. Scanning electron microscopy showed climbing fiber retrograde collaterals in the molecular layer. Asymmetric synaptic contacts of climbing fibers with Purkinje dendritic spines and stellate neuron dendrites were characterized by transmission electron microscopy. Correlative microscopy allowed us to obtain the basic three-dimensional morphological features of climbing fibers in several vertebrates and to show with more accuracy a higher degree of lateral collateralization of these fibers within the cerebellar cortex. The correlative microscopy approach provides new views in the cerebellar cortex information processing.

The central projection of cephalic mechanosensory axons in the haematophagous bug Triatoma infestans

The projections of mechanosensory hairs located on the dorsal and lateral head of the adult haematophagous bug Triatoma infestans were analyzed by means of cobalt filling. Axons run into the anterior and posterior tegumentary nerve and project through the brain to the ventral nerve cord. The fibres are small in diameter and run as a fascicle. Some branches run into suboesophageal and prothoracic centres; others run as far as to the mesothoracic ganglion. These sensory projections resemble that of wind-sensitive head hairs of the locust. The functional role of this sensory system in this species is discussed.

Electron microscopy of glial cells of the central nervous system in the crab Ucides cordatus

Invertebrate glial cells show a variety of morphologies depending on species and location. They have been classified according to relatively general morphological or functional criteria and also to their location. The present study was carried out to characterize the organization of glial cells and their processes in the zona fasciculata and in the protocerebral tract of the crab Ucides cordatus. We performed routine and cytochemical procedures for electron microscopy analysis. Semithin sections were observed at the light microscope. The Thiéry procedure indicated the presence of carbohydrates, particularly glycogen, in tissue and in cells. To better visualize the axonal ensheathment at the ultrastructural level, we employed a method to enhance the unsaturated fatty acids present in membranes. Our results showed that there are at least two types of glial cells in these nervous structures, a light one and a dark one. Most of the dark cell processes have been mentioned in the literature as extracellular matrix, but since they presented an enveloping membrane, glycogen and mitochondria - intact and with different degrees of disruption - they were considered to be glial cells in the present study. We assume that they correspond to the perineurial cells on the basis of their location. The light cells must correspond to the periaxonal cells. Some characteristics of the axons such as their organization, ensheathment and subcellular structures are also described

Ultrastructure of cranial nervs of rats inoculated with rabies virus

The V and VII cranial nerves of rats inoculated with rabies virus were studied by electron microscopy. The results were compared with the same cranial nerves of rats inoculated with rabies virus but vaccinated against the disease. The findings are those of axonal degeneration and intense demyelination of the nerves of the group of rats not vaccinated. The vaccinated rats showed some ultrastructural irrelevant alterations when compared with the other group. The degreee of ultrastructural alterations found in the group of rats not vaccinated suggests that in rabies severe damage of the cranial nerves occurs and that this may be closely related to the clinical picture of the disease (hydrophobia). Furthermore, as far as the authors know, this has not been considered in the classic descriptions of rabies and it is possible that an immunologic process may take part in the demyelination observed in the present study.

Quantitative analysis of the myelinated axons in the fasciculus gracilis and fasciculus cuneatus: light microscopy study

Fueron estudiados los fascículos grácil y cuneiforme de la médula espinal del Rattus norvegicus, mutante de la línea Wistar a nivel del primer y sexto segmentos cervicales, empleando la microscopía óptica, con la finalidad de evaluar el grado de mielinización para las edades cero, 1, 3, 5, 10, 15, 20, 30, 120 días postnatal. Se observó que el fascículo grácil se presenta al nacimiento prácticamente amielínico, con gran actividad de mielinización ocurriendo posterior al quinto día. A los 20 días, a nivel del primer segmento cervical, más de la mitad de la mielinización ya se completó en relación a la mielinación del animal adulto con 120 días. Al nacimiento, el fascículo cuneiforme es portador de fibras mielinizadas, manteniéndose siempre con mayor grado de mielinación que el fascículo grácil para cada edad evaluada. A los 20 días, el fascículo cuneiforme, a nivel del primer segmento cervical, se apresentó con más de 3/4 de la mielinización completa en relación al animal adulto de 120 días. En este estudio se observó que hay una gradiente de mielinización rostro-caudal, pues el grado de mielinización fue siempre mayor en el primer que en el sexto segmento cervical. Así, la mielinización de los fascículos grácil y cuneiforme de la médula espinal del Rattus norvegicus mutante de la línea Wistar, se presentó en su mayor parte, concordante con los resultados obtenidos por otros investigadores, demostrando que la mutación ocurrida en este animal, la cual se expresó bajo el fenotipo de piel glabra, no afectó de forma significativa el aspecto de la mielinización

Limits of variation of fiber distribution in the sural nerve of man

Although the sural nerve is the most extensively studied nerve in man, there is a dearth of data regarding the normal variations in the size-frequency distribution of axons in normal subjects; criteria for assessing the normality of a given individual are not available. Therefore, in everyday practice, the surgical pathologist may meet with difficulty in interpretit the biopsy of one particular individual, in whom the distribution is slightly different from the curves published. The object of this work is to detect the normal limits of variation in the distribution of diameters of muelinated and unmyelinated fibers in normal subjects and to establish the criteria that permit the calculated curves to be used in veryday clinical practice. Normal sural nerves of 19 patients were analyzed. Ages ranged between 18 months and 55 years. Morphometric analysis was performed with the Histoscan X automatic image processing analyzer, and , for statistical analysis, mixtures of lognormal distributions were fitted and tested with Pearson's statistics. Nerves of three diabetic patients were used for testing the method. They were clearly classified as abnormal. The curves, therefore, have been proven useful for everyday surgical pathology practice

Doença de Charcot-Marie-Tooth: estudo da biópsia do nervo sural em 41 pacientes / Charcot-Marie-Tooth disease: study of the sural nerve biopsy in 41 patients

Säo apresentados os resultados da biópsia do nervo sural à microscopia óptica e eletrônica (ME) em 41 pacientes com doença de Charcot-Marie-Tooth (CMT). Por estudos de neuroconduçäo prévios nove eram do tipo I e 32 do tipo II. No tipo I, todos tinham grande diminuiçäo do número de fibras, sendo os histogramas do tipo unimodal. Encontramos imagens de desmielinizaçäo, remielinizaçäo, formaçäo de bulbos de cebola e de regeneraçäo. Um paciente apresentava espessamento da bainha de mielina (atrofia axonal). No tipo II, sete pacientes näo apresentavam anomalia à microscopia, com histograma normal. Nos restantes havia discreta a intensa perda de fibras mielínicas principalmente as de grande calibre. Em cinco enfermos havia aumento do número de fibras devido a grande regeneraçäo. Alguns pacientes do tipo II apresentavam à ME imagens de pequenos bulbos de cebola e em um havia também atrofia axonal. Comparando com os dados clínicos e de neurconduçäo motora, no tipo I näo encontramos relaçäo entre a intensidade do quadro clínico e da perda de fibras mielínicas porém houve paralelismo da queda da neuroconduçäo motora e a diminuiçäo do número de fibras. No tipo II näo houve relaçäo entre o quadro clínico, a neuroconduçäo e os achados da biópsia nervosa

Poda selectiva de las proyecciones comisurales del colículo superior de la rata durante el desarrollo posnatal / Selective pruning of commisural projections in the superior colliculus of the rat during posnatal development

En el presente trabajo he estudiado los cambios que ocurren en proyecciones intercoliculares rata durante el desarrollo posnatal de la rata. El uso de la carbocianina DiI ha permitido marcar los axones comisurales del colículo superior. Durante el período postnatal temprano (P-1 a P-5), se observan proyecciones comisurales muy abundantes, que se distribuyen a gran parte del colículo superior contralateral. A partir del día 15 estas proyecciones se van reduciendo para adquirir, alrededor del día 30, el aspecto observado en el adulto. Existe, por lo tanto, un proceso de poda o eliminación selectiva de las proyecciones intercoliculares durante el desarrollo postnatal

Polineuropatia por triclorfon: registro de um caso com estudo eletrofisiológico e histopatológico do nervo sural / Polyneuropathy by trichlorfon: report of a case with electrophysiological and histopathological study of the sural nerve

É apresentado caso de paciente agricultor que, após uso inadequado de inseticida organofosforado (triclorfon), teve sinais e sintomas de intoxicaçäo aguda e, três meses após, desenvolveu quadro de polineuropatia sensitivo-motora. O exame eletroneuromiográfico revelou alteraçöes axonais e desmielinizantes difusas. O estudo do nervo sural, em cortes semi-finos, à ultramicroscopia e à técnica de fibras isoladas revelou degeneraçäo axonal e transformaçäo grnaular do axoplasma com perda dos neurofilamentos e neurotúbulos

Observation on the arrangement of axons according to diameter in the optic tract of the opossum Didelphus Marsupialis

The spectra of fiber sizes at different depths of the optic tract of the opossum didelphis marsupialis were examined by electron microscopy in order to test for correlations between the eventual location of axons and relevant developmental events. Frequency histograms showed 1) a predominant representation of medium-sized axons and the virtual exclusion of coarse fibers from the deepest portion of that pathway, and 2) a progressive increase in the poportion of thin axons from deep to superficial sites of the tract. These findings are discussed in terms of the view of the optic tract as a chronological map of axon arrival

Correlaçäo clínico-patológica em 78 biópsia de nervo sural / Sural nerve biopsy: clinical-pathologic correlation in 78 patients

As biópsias de nervos periféricos processadas com técnicas convencionais de parafina muitas vezes näo fornecem dados suficientes para a conclusäo diagnóstica. Se, no entanto, o nervo for processado para inclusäo em resina para cortes semi e ultrafinos e microdissecçäo de fibras, vários aspectos podem ser analisados inclusive por métodos quantitativos ou morfométricos. Estudamos o nervo sural de 78 pacientes biopsiados no Hospital Universitário Antonio Pedro, Niterói, utilizando essas técnicas e observamos que em 55 casos (70,5%) o diagnóstico anátomo-patológico foi conclusivo, em 11(14,1%) o nervo estava normal. Em 68 casos havia um diagnóstico clínico que foi confirmado em 49 casos, o que näo ocorreu nos 19 restantes pois, 8 tinham alteraçöes inespecíficas e 11 eram normais. Nos 10 casos em que a situaçäo nosológica näo foi estabelecida clinicamente, a biópsia foi conclusiva em 6, exibia alteraçöes inespecíficas em 4, sendo normal em 1 caso. A conclusäo diagnóstica na maioria dos nossos casos só foi possível porque, além dos dados clínicos fornecidos, todos os nervos foram processados para inclusäo em resina

Neuropatia pela amiodarona: estudo clínico-patológico de dois casos / Amiodarone neuropathy: clinico-pathological study of 2 cases

Dois pacientes, uma mulher de 40 anos e um homem de 75 anos, apresentavam polineurite sensitivo-motora, cujo único antecedentes era o uso de amiodarona há 4 e 6 anos respectivamente. O ENMG revelou quadro neuropático tipo axonal. A biópsia do nervo sural mostrou rarefaçäo axonal, mielínica e amielínica, bem como inclusöes lamelares osmiofílicas nas células de Schwann e no endotélio venular. Com a retirada da amiodarona houve regressäo da polineurite. A semelhança do que foi descrito na neuropatia pelo maleato de perhexiline e pela cloroquina, a amiodarona constitui constitui importante fator de induçäo de neurolipidose medicamentosa

Alteraçöes da junçäo neuromuscular em miopatias experimentais no camundongo / Neuromuscular junction changes in experimental myopathies in mice

As alteraçöes morfológicas observadas em junçäo neuromuscular de dois modelos de miopatia em camundongos säo estudadas por métodos histoquímicos para demonstrar atividade da enzima acetilcolinesterase e por microscopia eletrônica. Em ambas as situaçöes os resultados obtidos säo similares, indicando que a junçäo neuromuscular permanece intacta mesmo quando a fibra que inerva está sofrendo necrose. Em fibras musculares regeneradas há acentuada simplificaçäo das pregas pós-sinápticas, com reduçäo de até 50% dos valores normais, comprovado por estudos morfométricos. A ausência de reprecussöes fisiológicas ou clinicas detectáveis nesses modelos, apesar da significancia hipotrfia da membrana pós-sináptica, sugere que a exuberante quantidade de pregas pós-sinápticas normalmente encontradas nas junçöes mioneurais pode representar mecanismo anatômico de segurança na transmissäo química neuromuscular

How many channels should there be in a gap junction?

La resistencia de las electrotónicas (uniones comunicantes) formadas entre los axones laterales gigantes del cangrejo de río ha sido medicda en preparaciones acopladas y desacopladas. Por otra parte el número de partículas sinápticas se ha contado en ambas condiciones. De estos dos grupos de datos se puede concluir que las partículas, agrupadas en placas, que se observan mediante criofractura representan canales comunicantes y que estos están abiertos. Basándonos en estos datos y en resultados publicados obtenidos en pares aislados de células acopladas se ha encontrado que existe una relación inversa entre la resistencia de entrada de las células y el número estimado de canales comunicantes abiertos entre ellas. Esta relación se aplica tanto a células excitables como a las no excitables