Fine structure ofPeriplaneta americana neurons in long-term culture

Beadle, David J.; Hicks, David; Middleton, C.

doi:10.1007/bf01262427

Cited by 43 publications

(10 citation statements)

References 26 publications

(32 reference statements)

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“…4). As both neurons and glial cells can be severely damaged during the culturing procedure, many of them die and disintegrate (Beadle et al, 1982(Beadle et al, , 1987; this study). Therefore viability testing is essential to distinguish HRP-negative glial cells from membraneless remnants of dead cells.…”

Section: Microscopy Research and Techniquementioning

confidence: 96%

“…Nevertheless, the morphology of glial cells from adult cockroaches (Howes et al, 1989), pupal honeybees (Gascuel and Masson, 1991), and locust embryos ( Vanhems and Delbos, 1987) has been studied in vitro and a number of functional studies revealed that insect glial cells express high affinity transporters for L-glutamate, GABA, and histamine (Campos-Ortega, 1974;Borycz et al, 2002;Soustelle et al, 2002, Freeman et al, 2003, respond to neuronal transmitters (Giles and Usherwood, 1985;Leitch et al, 1993;Schofield and Treherne, 1985) and may require neuron-derived trophic signals for survival and differentiation (Bergmann et al, 2002;Hidalgo et al, 2001;Sen et al, 2004). A prerequisite for in vitro studies on primary cell cultures from insect nervous systems is the reliable distinction of glial and neuronal cell bodies, which is especially problematic in fresh cell cultures, where both cell types assume spherical shapes and lack characteristic processes (Beadle et al, 1982(Beadle et al, , 1987. A simple and widely used method to identify glial cells in histological studies is based on the absence of antihorseradish peroxidase (HRP) immunoreactivity, which labels the pan-neuronally expressed surface protein Nervana (Jan and Jan, 1982;Sun and Salvaterra, 1995a,b) and this method has recently been adopted to identify cultured glia cells (Loesel et al, 2006).…”

Section: Introductionmentioning

confidence: 98%

“…Various studies reported that glial cells are especially sensitive to mechanical dissociation of central nervous tissue leading to a low abundance of surviving glial cells in primary cell cultures (Beadle et al, 1982;LeviMontalcini et al, 1973;Vanhems and Delbos, 1987). Nevertheless, the morphology of glial cells from adult cockroaches (Howes et al, 1989), pupal honeybees (Gascuel and Masson, 1991), and locust embryos ( Vanhems and Delbos, 1987) has been studied in vitro and a number of functional studies revealed that insect glial cells express high affinity transporters for L-glutamate, GABA, and histamine (Campos-Ortega, 1974;Borycz et al, 2002;Soustelle et al, 2002, Freeman et al, 2003, respond to neuronal transmitters (Giles and Usherwood, 1985;Leitch et al, 1993;Schofield and Treherne, 1985) and may require neuron-derived trophic signals for survival and differentiation (Bergmann et al, 2002;Hidalgo et al, 2001;Sen et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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Recognition, presence, and survival of locust central nervous glia in situ and in vitro

Gocht

Wagner

Heinrich

2008

Microscopy Res & Technique

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Insect glial cells serve functions for the formation, maintenance, and performance of the central nervous system in ways similar to their vertebrate counterparts. Characterization of physiological mechanisms that underlie the roles of glia in invertebrates is largely incomplete, partly due to the lack of markers that universally label all types of glia throughout all developmental stages in various species. Studies on primary cell cultures from brains of Locusta migratoria demonstrated that the absence of anti-HRP immunoreactivity, which has previously been used to identify glial cells in undissociated brains, can also serve as a reliable glial marker in vitro, but only in combination with a viability test. As cytoplasmic membranes of cultured cells are prone to degradation when they lose viability, only cells that are both anti-HRP immunonegative and viable should be regarded as glial cells, whereas the lack of anti-HRP immunoreactivity alone is not sufficient. Cell viability can be assessed by the pattern of nuclear staining with DAPI (4',6-diamidino-2-phenylindole), a convenient, sensitive labeling method that can be used in combination with other immunocytochemical cellular markers. We determined the glia-to-neuron ratio in central brains of fourth nymphal stage of Locusta migratoria to be 1:2 both in situ and in dissociated primary cell cultures. Analysis of primary cell cultures revealed a progressive reduction of glial cells and indicated that dead cells detach from the substrate and vanish from the analysis. Such changes in the composition of cell cultures should be considered in future physiological studies on cell cultures from insect nervous systems.

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Section: Microscopy Research and Techniquementioning

confidence: 96%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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Recognition, presence, and survival of locust central nervous glia in situ and in vitro

Gocht

Wagner

Heinrich

2008

Microscopy Res & Technique

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“…By following the method of Beadle et al (1982), APRs were fixed for 1 hour at 4°C in 2% gluteraldehyde (grade I, 25% aqueous; Sigma) in 0.1 M cacodylate buffer (pH 7.2). The interval between removal from the ganglion and fixation was 30 -60 minutes, depending on the number of cells to be processed.…”

Section: Preparation Of Aprs For Transmission Emmentioning

confidence: 99%

Steroid‐triggered programmed cell death of a motoneuron is autophagic and involves structural changes in mitochondria

Kinch

Hoffman

Rodrigues

et al. 2003

J of Comparative Neurology

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Neuronal death occurs during normal development and disease and can be regulated by steroid hormones. In the hawkmoth, Manduca sexta, individual accessory planta retractor (APR) motoneurons undergo a segment-specific pattern of programmed cell death (PCD) at pupation that is triggered directly and cell autonomously by the steroid hormone 20-hydroxyecdysone (20E). APRs from abdominal segment six [APR(6)s] die by 48 hours after pupal ecdysis (PE; entry into the pupal stage), whereas APR(4)s survive until adulthood. Cell culture experiments showed previously that 20E acts directly on APRs to trigger PCD, with intrinsic segmental identity determining which APRs die. The APR(6) death pathway includes caspase activation and loss of mitochondrial function. We used transmission electron microscopy to investigate the ultrastructure of APR somata before and during PCD. APR(4)s showed normal ultrastructure at all stages examined, as did APR(6)s until approximately stage PE. During APR(6) death, there was massive accumulation of autophagic bodies and vacuoles, mitochondria became ultracondensed and aggregated into compact clusters, and ribosomes aggregated in large blocks. Nuclear ultrastructure remained normal, without chromatin condensation, until the nuclear envelope fragmented late in the death process. Light microscopic immunocytochemistry showed that dying APR(6)s were TUNEL-positive, which is diagnostic of fragmented DNA. These observations indicate that the steroid-induced, caspase-dependent, cell-autonomous PCD of APR(6)s is autophagic, not apoptotic, and support an early role for mitochondrial alterations during PCD. This system permits the study of neuronal death in response to its bona fide developmental signal, the rise in a steroid hormone.

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“…Since many years also insect neurons have been used in cell culture [2][3][4][5][6][7][8][9]. The cells in those cultures are often unidentified, thereby loosing the advantages of the identified neuron concept.…”

Section: Introductionmentioning

confidence: 99%

Thoracic Interneurons, Motorneurons and Sensory Neurons of Locusta Migratoria (Insecta: Orthoptera) in Primary Cell Culture

Pfahlert¹,

Lakes‐Harlan²

2008

TOENTOJ

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Abstract:The aim of this study was to establish a cell culture system with identified classes of locust neurons (interneurons, motorneurons and sensory neurons). The cells belonging to the different classes were distinguished in cell culture by vital dyes, which had been applied to the neurons in situ. From the various dyes tested fluorescent marked dextrans (10.000MW) gave the best results. The cells survived for up to 28 days in culture and approx. half of the cells grew processes, except of the sensory neurons, which never formed any processes. The different neurons were comparatively investigated, e.g. with immunhistochemistry: 86% of motorneuron were glutamate immunoreactive and 50% of the interneurons exhibited GABA-like immunoreactivity. The cells had resting potentials between -20 and -60mV and did not show spontanous action potentials. Action potentials could be elicited by current injection in 8% of interneurons and 26% of motorneurons, but not in sensory neurons. The vital marking of cells allowed to study distinct neurons in cell culture and to compare their morphology and physiology.

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Fine structure ofPeriplaneta americana neurons in long-term culture

Cited by 43 publications

References 26 publications

Recognition, presence, and survival of locust central nervous glia in situ and in vitro

Recognition, presence, and survival of locust central nervous glia in situ and in vitro

Steroid‐triggered programmed cell death of a motoneuron is autophagic and involves structural changes in mitochondria

Thoracic Interneurons, Motorneurons and Sensory Neurons of Locusta Migratoria (Insecta: Orthoptera) in Primary Cell Culture

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