Tissue printed cells from teleost electrosensory and cerebellar structures

A modification of the tissue printing technique was used to acutely isolate and culture cells from the electrosensory lateral line lobe (ELL), corpus cerebelli (CCb), and eminentia granularis pars posterior (EGp) of the adult weakly electric fish, Apteronotus leptorhynchus. Cells were isolated without the use of proteolytic enzymes and tissue printed as a monolayer onto glass coverslips through centrifugation in the presence of a medium designed to preserve cell structure. Tissue printed cells were reliably distributed in an organotypic fashion that allowed for the identification of anatomical boundaries between the ELL and cerebellar regions, distinct sensory maps in the ELL, and specific cell laminae. Many cells were isolated with an excellent preservation of soma-dendritic structure, permitting direct identification of all electrosensory cell classes according to morphological or immunocytochemical criteria. Several classes of glial cells were isolated, including small diameter microglia and the complex arborizations of oligodendrocytes. A plexus of fine processes were often isolated in conjunction with cell somata and dendrites, potentially preserving synaptic contacts in vitro. In particular, immunolabel for γ-aminobutyric acid (GABA) revealed a previously unrecognized network of GABAergic axonal processes in the CCb and EGp granule cell body and molecular layers. Tissue printed cells were readily maintained with an organotypic distribution of glial and neuronal elements for up to 27 days in culture. This procedure will allow for the isolation of electrosensory cells from adult central nervous system for electrophysiological analyses of membrane properties or synaptic interactions between identified cells. J. Comp. Neurol. 386:277–292, 1997. © 1997 Wiley-Liss, Inc.     

Spatial distribution of ω-agatoxin IVA binding sites in mouse brain slices

A peptide toxin derived from funnel-web spider venom, ω-agatoxin IVA, blocks voltage-sensitive calcium channels. Many pharmacological and electrophysiological studies have shown that these channels are widely distributed in both the central nervous system (CNS) and neuromuscular junctions. However, a direct morphological demonstration of the binding sites of this toxin is still lacking. To identify which cells have the binding sites, a biologically active, biotin-conjugated ω-agatoxin IVA was applied to mouse cerebellar and hippocampal slices. Confocal microscopy revealed that ω-agatoxin IVA binding sites were distributed on the somata of Purkinje cells, cerebellar granule cells and interneurons, as well as on the dendrites of Purkinje cells. In the hippocampus, the binding sites were localized on the somata of pyramidal cells of the CA1–CA4 region and on the somata of granule cells in the dentate gyrus. A sequential competitive reaction confirmed the specificity of the binding in the cerebellum and CA1 pyramidal cells, and also suggested a difference in the binding affinity between CA1 and CA3 pyramidal cells. Since a high concentration of ω-agatoxin IVA (2 μM) was needed for the present study, the ω-agatoxin IVA binding sites presented in this study may represent “P-type” and “Q-type” calcium channels. © 1995 Wiley-Liss, Inc.     

Mormyrid electrosensory lobe in vitro: Morphology of cells and circuits

The electrosensory lobe (ELL) of mormyrid electric fish is a cerebellum-like brainstem structure that receives the primary afferent fibers from electroreceptors in the skin. The ELL and similar sensory structures in other fish receive extensive input from other central sources in addition to the peripheral input. The responses to some of these central inputs are adaptive and serve to minimize the effects of predictable sensory inputs. Understanding the interaction between peripheral and central inputs to the mormyrid ELL requires knowledge of its functional circuitry, and this paper examines this circuitry in the in vitro slice preparation and describes the axonal and dendritic morphology of major ELL cell types based on intracellular labeling with biocytin. The cells described include medium ganglion cells, large ganglion cells, large fusiform cells, thick-smooth dendrite cells, small fusiform cells, granule cells, and primary afferent fibers. The medium ganglion cells are Purkinje-like interneurons that terminate on the two types of efferent cells, i.e., large ganglion and large fusiform cells, as well as on each other. These medium ganglion cells fall into two morphologically distinct types based on the distributions of basal dendrites and axons. These distributions suggest hypotheses about the basic circuit of the ELL that have important functional consequences, such as enhancement of contrast between “on” elements that are excited by increased afferent activity and “off” elements that are inhibited. J. Comp. Neurol. 404:359–374, 1999. © 1999 Wiley-Liss, Inc.     

Immunocytochemical identification of cell types in the mormyrid electrosensory lobe

The electrosensory lobes (ELLs) of mormyrid and gymnotid fish are useful sites for studying plasticity and descending control of sensory processing. This study used immunocytochemistry to examine the functional circuitry of the mormyrid ELL. We used antibodies against the following proteins and amino acids: the neurotransmitters glutamate and gamma-aminobutyric acid (GABA); the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD); GABA transporter 1; the anchoring protein for GABA and glycine receptors, gephyrin; the calcium binding proteins calbindin and calretinin; the NR1 subunit of the N-methyl-D-aspartate glutamate receptor; the metabotropic glutamate receptors mGluR1alpha, mGluR2/3, and mGluR5; and the intracellular signaling molecules calcineurin, calcium calmodulin kinase IIalpha (CAMKIIalpha) and the receptor for inositol triphosphate (IP3R1alpha). Selective staining allowed for identification of new cell types including a deep granular layer cell that relays sensory information from primary afferent fibers to higher order cells of ELLS. Selective staining also allowed for estimates of relative numbers of different cell types. Dendritic staining of Purkinje-like medium ganglion cells with antibodies against metabotropic glutamate receptors and calcineurin suggests hypotheses concerning mechanisms of the previously demonstrated synaptic plasticity in these cells. Finally, several cell types including the above-mentioned granular cells, thick-smooth dendrite cells, and large multipolar cells of the intermediate layer were present in the two zones of ELL that receive input from mormyromast electroreceptors but were absent in the zone of ELL that receives input from ampullary electroreceptors, indicating markedly different processing for these two types of input. J. Comp. Neurol. 483:124-142, 2005. (c) 2005 Wiley-Liss, Inc.     

ω-Conotoxin reduces facilitation of transmitter release at the frog neuromuscular junction

We have examined the effects of the peptide toxin ω-conotoxin GVIA (ω-CgTx), a known calcium channel blocker, on stimulation-induced changes in end-plate potential (EPP) amplitude at the frog neuromuscular junction. We found that the addition of this toxin in submicromolar concentrations reduced both the control EPP amplitude and the increase in EPP amplitude that normally occurs during repetitive stimulation under low quantal conditions. These effects of ω-CgTx developed slowly following its addition to the bathing solution, were concentration-dependent and were essentially irreversible. The effects of ω-CgTx appeared to result from reductions in the facilitation and augmentation components of stimulation-induced increases in release. While the effects of ω-CgTx on EPP amplitude could be reversed by increasing the extracellular concentration of Ca²⁺, we were unable to reverse the effects of the toxin on stimulation-induced increases in EPP amplitude. Thus it appears that ω-CgTx has a dual effect on neuromuscular transmission, perhaps by acting at two different presynaptic sites.     

Distribution of noradrenaline-immunoreactivity in the brain of the mormyrid teleost Gnathonemus petersii

The distribution of noradrenaline-immunoreactivity in the brain of the mormyrid fish Gnathonemus petersii was studied in order to evaluate the noradrenergic innervation of a number of specialized mormyrid brain regions, including electrosensory centers and a gigantocerebellum. Noradrenaline-immunoreactive (NAi) neurons occur in the hypothalamic paraventricular organ (PVO), the locus coeruleus, and the caudal rhombencephalon. In the PVO, NAi cerebrospinal fluid (CSF)-contacting neurons are located in the same regions where dopamine- and serotonin-containing CSF contacting neurons occur. The locus coeruleus consists, on each side, of at least 30 rather large NAi neurons with ventrolaterally directed dendrites and dorsolaterally coursing axons. In the caudal rhombencephalon, NAi neurons are located in the transition region between the ventromedial motor zone and the dorsolateral sensory zone. The density of NAi fibers is very high in the efferent tract of the locus coeruleus, the medial forebrain bundle, and two telencephalic, one preoptic, and one rhombencephalic subependymal axonal plexus. A marked NAi innervation is present in the dorsomedial and ventral telencephalon, the preoptic region, periventricular hypothalamic and thalamic regions, the midbrain tectum, cerebellar granular layers, the electrosensory lateral line lobe, the rhombencephalic transition region between the sensory and motor zones, and the area postrema. Other regions are more sparsely innervated by NAi fibers, but regions completely devoid of NAi fibers were not observed. Interestingly, NAi fibers form large club endings in some subdivisions of the precerebellar nucleus lateralis valvulae, and parallel fibers in the cerebellar granular layer. Comparison with the distribution of NAi or dopamine-β-hydroxylase-immunoreactivity in other species shows that all teleosts studied to date have noradrenergic cells in the locus coeruleus and the caudal rhombencephalon. However, NAi CSF-contacting PVO cells have been described only in the teleost Gnathonemus petersii and the lizard Gekko gecko (Smeets and Steinbusch: J. Comp. Neurol. 285:453–466, 89). It is possible that they might pick up catecholamines as well as serotonin from the CSF, into which monoamines might be released by telencephalic and preoptic subependymal axonal plexuses. © 1993 Wiley-Liss, Inc.     

Acutely isolated and cultured cells from the electrosensory lateral line lobe of a gymnotiform teleost

The present study established the morphological and immunocytochemical criteria necessary to identify neuronal and nonneuronal cells after dissociating select regions of the medullary electrosensory lateral line lobe of adult weakly electric fish (Apteronotus Zeptorhynchus). Cells dissociated from the pyramidal cell body layers of the centromedial and lateral segments exhibited similar characteristics in the acutely dissociated preparation and up to 14 days in culture. Basilar and nonbasilar pyramidal cells were tentatively identified according to a bipolar or monopolar process extension, and polymorphic cells by the extension of three or more processes and positive immunoreactivity for gamma-aminobutyric acid. Nonneuronal cells were identified by the pattern of process arborization and positive immunolabel for gamma-aminobutyric acid or glial fibrillary acidic protein. Neuronal cells increased in total number over the first 4 days and could appear for the first time on any day in culture. Individual pyramidal cells could maintain their morphology from the time of dissociation and over several days in culture. Pyramidal cell processes were phenotypically similar to apical and basal dendrites found in situ but were reduced in size and in the degree of process branching. These results indicate that dissociated adult apteronotid neurons can maintain a morphology sufficiently similar to that found in situ as to allow tentative identification, opening up a wide range of possibilities for studying the electrophysiological and regenerative properties of electrosensory neurons. © 1995 Wiley-Liss, Inc.     

Presynaptic localization of ω-conotoxin-sensitive calcium channels at the frog neuromuscular junction

By the use of anti-ω-CTx antibodies in indirect immunofluorescence we demonstrated the presence of ω-CTx binding sites in the presynaptic compartment of frog nerve-muscle preparations. The images we obtained indicate that ω-CTx-sensitive channels are clustered at discrete sites corresponding in distribution to active zones.     

Autoradiographic visualization of a calcium channel antagonist, [I]ω-conotoxin GVIA, binding site in the brains of normal and cerebellar mutant mice (pcd andweaver)

An in vitro autoradiographic technique has been used to localize [¹²⁵I]ω-conotoxin GVIA binding sites in the brains of normal and cerebellar mutant mice. In the brains of normal mice, the highest densities of binding sites were observed at glomeruli of the olfactory bulb, cerebral cortex, caudate nucleus-putamen, hippocampus, and the nucleus of the solitary tract. Moderate densities of the silver grains occured on the granular layer of the olfactory bulb, the molecular layer of the dentate gyrus, the molecular layer of the cerebellum, and the cochlear nucleus. No specific binding appeared in the white matter or the deep nucleus of the cerebellum, the corpus callosum, the internal capsule and the external plexiform layer of the olfactory bulb. Autoradiographic studies of the cerebella of Purkinje cell degeneration (pcd) mice showed that the distribution of binding sites on the molecular layer of the cerebellum are not affected by the degeneration of Purkinje cells. However, only background levels of the silver grains occured on the cerebella of agranularweaver mutant mice, suggesting that the receptor for ω-conotoxin GVIA in the cerebellum are predominantly distributed on the parellel fibers of granule cells.     

Different ω-conotoxins mark the development of Swiss Webster mouse cortex suggesting N-Type voltage sensitive calcium channel subtypes

ω-GVIA conotoxin has been used to mark presynaptic N-type voltage sensitive calcium channels (VSCC).³,¹³,¹⁹,^21–23 Litzinger et al.⁹ used ω-conotoxin binding to describe a critical period of neurodevelopment in Swiss Webster mice between postnatal days (PND) 11 and 14, which appears to be important to the initiation of proper final development of the central nervous system. In this study, we compare how three different ω-conotoxins (i.e. GVIA from Conus geographus, MVIIA from Conus magus, and RVIA from Conus radiatus) mark N-type VSCC during this critical period in Swiss Webster mouse cortex. ¹²⁵I-GVIA was bound to Swiss Webster mouse cortex synaptosomal membrane fractions at postnatal days 8 and 14. ¹²⁵I-GVIA binding displacement curves were obtained by incubating membranes with increasing concentrations of unlabeled GVIA, MVIIA, and RVIA. Displacement curves and IC₅₀ were calculated for each of these three ω-conotoxins, and then compared. At PND 14, GVIA, MVIIA and RVIA were able to displace greater than 95% of ¹²⁵I-GVIA binding. At PND 8, however, MVIIA was only able to displace 83% of ¹²⁵I-GVIA binding, and RVIA was only able to block 84%. The IC₁₅₀ does not appear to change significantly during this period of development for any of the ω-conotoxins. The inability of MVIIA and RVIA to completely block ¹²⁵I-GVIA binding in pre-critical period Swiss Webster cortex denotes an alteration in the composition of N-type VSCC binding sites. With this data, we have suggested the presence of subtypes of the N-type VSCC in the cortex of pre-critical period Swiss Webster mouse.     

Distribution of various calcium channel α1 subunits in murine DRG neurons and antinociceptive effect of ω-conotoxin SVIB in mice

Immunohistological study revealed the differential localization of subtypes of voltage-dependent calcium channels in the dorsal root ganglion neurons. Intrathecal injection of ω-conotoxin SVIB, an analogue of ω-conotoxin GVIA, which acts on N-type voltage-dependent calcium channels, significantly shortened the licking time in the late phase of a formalin test.     

Suppression of hippocampal synaptic transmission by the spider toxin ω-agatoxin-IV-A

Effects of ω-agatoxin-IV-A (AGTX) on synaptic transmissions were examined in thin transverse sections of the guinea pig hippocampus. AGTX suppressed, in a dose-dependent manner, field potentials elicited by mossy fiber stimulation in CA3 pyramidal cell layer. AGTX also suppressed field potentials elicited by perforant fiber stimulation in granular layer without marked changes in antidromic responses. These results suggest that Ca²⁺ entry into presynaptic terminals via P-like calcium channels is at least partly responsible for release of glutamate.     

A prominent soma-dendritic distribution of Kv3.3 K+ channels in electrosensory and cerebellar neurons.

The expression pattern and subcellular distribution of a teleost homologue of the mammalian Kv3.3 potassium channel, AptKv3.3, was examined in the electrosensory lateral line lobe (ELL) and two cerebellar lobes in the hindbrain of the weakly electric gymnotiform Apteronotus leptorhynchus. AptKv3.3 expression was brain specific, with the highest level of expression in the cerebellum and 56% relative expression in the ELL. In situ hybridization revealed that AptKv3.3 mRNA was present in virtually all cell classes in the ELL as well as in the cerebellar lobes eminentia granularis pars posterior (EGp) and corpus cerebellum (CCb). Immunocytochemistry indicated a distribution of AptKv3.3 channels over the entire soma-dendritic axis of ELL pyramidal, granule, and polymorphic cells and over the soma and at least proximal dendrites (100 microm) of multipolar cells and neurons of the ventral molecular layer. AptKv3.3 immunolabel was present at the soma of cerebellar granule, golgi, eurydendroid, and CCb Purkinje cells, with an equally intense label throughout the dendrites of CCb Purkinje cells and EGp eurydendroid cells. Immunolabel was virtually absent in afferent or efferent axon tracts of the ELL but was detected on climbing fiber axons and on the axons and putative terminal boutons of CCb Purkinje cells. These data reveal a prominent soma-dendritic distribution of AptKv3.3 K+ channels in both principal output and local circuit neurons, a pattern that is distinct from the soma-axonal distribution that characterizes all other Kv3 K+ channels examined to date. The widespread distribution of AptKv3.3 immunolabel in electrosensory cells implies an important role in several aspects of signal processing.     

Spermine does not compete with ω-conotoxin GVIA in the stratum radiatum of the hippocampal slice

The effect of spermine (Spm) and of ω-conotoxin GVIA (CTX) on the population excitatory postsynaptic potentials (pEPSP) in stratum radiatum of the CA1 area were compared. CTX decreased irreversibly the initial slope of pEPSP by 57%. Spm produced a maximum inhibition of 85% with an apparent dissociation constant of 0.85 mM and a maximum Hill coefficient larger than 3. The effect of Spm was mostly reversible. Preincubation with Spm did not protect the slice from the irreversible effect of CTX suggesting that they interact with different sites. Since CTX and Spm inhibited pEPSPs with very different affinities and reversibilities a kinetic model was developed to compare their effects. This model relates the inhibitors' binding to presynaptic voltage-activated Ca²⁺ channels (VACC) with inhibition of pEPSP. The model suggest that: all CTX and Spm effects can be explained by inhibition of VACC. Spm and CTX do not compete for the same site. CTX inhibits 20% (N-type) and Spm 40% of channels (probably the Q-type). More than three Spm molecules bind per one channel molecule, while one CTX is sufficient to inhibit channel function. The model also illustrates that the inhibitor concentration–pEPSP inhibition curves display a Hill coefficient similar to that for inhibitor binding.     

Immunohistochemical and electrophysiological evidence for ω-conotoxin-sensitive calcium channels in unmyelinated C-fibres of biopsied human sural nerve

In vitro electrophysiological measurements of Ca²⁺ potentials in human sural nerve fascicles revealed that Ca²⁺ conductances might be present on unmyelinated C-fibres. Furthermore, these Ca²⁺ potentials were partially blocked by ω-conotoxin, a calcium antagonist for the N-type Ca²⁺ channels. Therefore, immunohistochemical staining with indirect immunofluorescent ω-conotoxin GVIA was used to localize N-type Ca²⁺ channels in intact and in enzymatically dissociated human sural nerve fascicles. Densities of toxin binding sites were highly heterogeneous throughout the different nerve fascicles investigated and putative N-type Ca²⁺ channels were localized in about 20% of the unmyelinated C-fibres. Myelinating Schwann cells as well as enzymatically demyelinated axons displayed no specific binding indicating the absence of N-type Ca²⁺ channels.     

γ‐Aminobutyric acid‐ρ expression in ependymal glial cells of the mouse cerebellum

The ependymal glial cells (EGCs) from the periventricular zone of the cerebellum were studied to determine their distribution and the functional properties of their γ‐aminobutyric acid type A (GABA_A) receptors. EGCs were identified by the presence of ciliated structures on their ventricular surface and their expression of glial fibrillary acidic protein (GFAP). Interestingly, diverse cell types, including neurons, astrocytes, and other types of glia, were identified in the subventricular zone by their current profiles. Electron microscopy showed ciliated cells and myelinated axons in this zone, but we found no collateral connections to suggest the presence of functional synapses. GABA‐mediated currents were recorded from EGCs in cerebellar slices from postnatal days 13 to 35 (PN13–PN35). These currents were blocked by TPMPA (a highly specific GABA_Aρ subunit antagonist) and bicuculline (a selective antagonist for classic GABA_A receptors). Pentobarbital failed to modulate GABA_A‐mediated currents despite the expression of GABAα1 and GABAγ2 subunits. In situ hybridization, RT‐PCR, and immunofluorescence studies confirmed GABAρ1 expression in EGCs of the cerebellum. We conclude that cerebellar EGCs express GABAρ1, which is functionally involved in GABA_A receptor‐mediated responses that are unique among glial cells of the brain. © 2013 Wiley Periodicals, Inc.     

Serotoninergic neurons in the mormyrid brain and their projection to the preelectromotor and primary electrosensory centers: immunohistochemical study

Serotonin-containing neurons in the brain of the weak-electric fish Gnathonemus petersii (mormyridae, teleostei) were studied with the aid of immunohistochemical labeling. Study of the central serotoninergic innervation was focused on the structures subserving the command of the electric organ and the first central relay of the electrosensory system. In the midline raphe nuclei, serotoninergic neurons formed a column that stretched from the ventral caudal medulla to the dorsal midbrain, ending caudal to the cerebellar peduncle. In the dorsal tegmentum, serotoninergic neurons were found bilaterally at the anterior margin of the decussation of the lateral lemniscus. Labeled neurons were also present bilaterally immediately anterior to the cerebellar peduncle and also in the pretectal region. In the hypothalamus, many serotoninergic neurons were in contact with the ventricular wall, and a few were present in the preoptic area. This distribution of serotoninergic cell bodies showed many similarities to that in other fish and higher vertebrates but lacked the lateral spread of the serotoninergic raphe system found in the midbrain tegmentum in mammals. Labeled fibers were found in both the preelectromotor medullary relay nucleus and the electromotor command nucleus. These serotoninergic projections were traced to the posterior raphe. Serotoninergic fibers also formed a dense network in the cortex and in the nucleus of the electrosensory lobe, both of which receive primary input from electroreceptors. These results suggest that serotonin may have a role in the modulation of the intrinsic, rhythmic electromotor command and in the gating of electrosensory input.     

Neuroprotective action of a novel compound–M50463–in primary cultured neurons

The neuroprotective effects of a novel synthetic compound, M50463, have been determined by using embryonic rat neocortical neurons in various culture conditions. M50463 was initially characterized as a potent specific ligand for a voltage-dependent sodium channel by radioligand binding studies. In fact, M50463 inhibited neuronal cell death induced by veratrine and inhibited an increase of the intracellular calcium level in neurons evoked by veratrine. In addition to such expected effects, M50463 had the ability to prevent glutamate neurotoxicity, to promote the neuronal survival in serum-deprived medium and to prevent nitric oxide-induced neurotoxicity. These results suggested that M50463 is not a simple sodium channel blocker, but a neuroprotective agent which has some crucial mechanism of action on neuronal death occurring in various situations, and it is a novel, innovative candidate for neuroprotective therapy for various neurodegenerative disorders.