GABA Immunoreactivity Research Articles

Immunocytochemical evidence for SNARE protein‐dependent transmitter release from guinea pig horizontal cells

Horizontal cells are lateral interneurons that participate in visual processing in the outer retina but the cellular mechanisms underlying transmitter release from these cells are not fully understood. In non-mammalian horizontal cells, GABA release has been shown to occur by a non-vesicular mechanism. However, recent evidence in mammalian horizontal cells favors a vesicular mechanism as they lack plasmalemmal GABA transporters and some soluble NSF attachment protein receptor (SNARE) core proteins have been identified in rodent horizontal cells. Moreover, immunoreactivity for GABA and the molecular machinery to synthesize GABA have been found in guinea pig horizontal cells, suggesting that if components of the SNARE complex are expressed they could contribute to the vesicular release of GABA. In this study we investigated whether these vesicular and synaptic proteins are expressed by guinea pig horizontal cells using immunohistochemistry with well-characterized antibodies to evaluate their cellular distribution. Components of synaptic vesicles including vesicular GABA transporter, synapsin I and synaptic vesicle protein 2A were localized to horizontal cell processes and endings, along with the SNARE core complex proteins, syntaxin-1a, syntaxin-4 and synaptosomal-associated protein 25 (SNAP-25). Complexin I/II, a cytosolic protein that stabilizes the activated SNARE fusion core, strongly immunostained horizontal cell soma and processes. In addition, the vesicular Ca(2+)-sensor, synaptotagmin-2, which is essential for Ca(2+)-mediated vesicular release, was also localized to horizontal cell processes and somata. These morphological findings from guinea pig horizontal cells suggest that mammalian horizontal cells have the capacity to utilize a regulated Ca(2+)-dependent vesicular pathway to release neurotransmitter, and that this mechanism may be shared among many mammalian species.

Allatostatin immunoreactivity in the honeybee brain

Information transmission and processing in the brain is achieved through a small family of chemical neurotransmitters and neuromodulators and a very large family of neuropeptides. In order to understand neural networks in the brain it will be necessary, therefore, to understand the connectivity, morphology, and distribution of peptidergic neurons, and to elucidate their function in the brain. In this study we characterize the distribution of substances related to Dip-allatostatin I in the honeybee brain, which belongs to the allatostatin-A (AST) peptide family sharing the conserved c-terminal sequence -YXFGL-NH(2). We found about 500 AST-immunoreactive (ASTir) neurons in the brain, scattered in 18 groups that varied in their precise location across individuals. Almost all areas of the brain were innervated by ASTir fibers. Most ASTir neurites formed networks within functionally distinct areas, e.g., the antennal lobes, the mushroom bodies, or the optic lobes, indicating local functions of the peptide. A small number of very large neurons had widespread arborizations and neurites were found in the corpora cardiaca and in the cervical connectives, suggesting that AST also has global functions. We double-stained AST and GABA and found that a subset of ASTir neurons were GABA-immunoreactive (GABAir). Double staining AST with backfills of olfactory receptor neurons or mass fills of neurons in the antennal lobes and in the mushroom bodies allowed a more fine-grained description of ASTir networks. Together, this first comprehensive description of AST in the bee brain suggests a diverse functional role of AST, including local and global computational tasks.

Allatostatin immunoreactivity in the honeybee brain

AbstractInformation transmission and processing in the brain is achieved through a small family of chemical neurotransmitters and neuromodulators and a very large family of neuropeptides. In order to understand neural networks in the brain it will be necessary, therefore, to understand the connectivity, morphology, and distribution of peptidergic neurons, and to elucidate their function in the brain. In this study we characterize the distribution of substances related to Dip‐allatostatin I in the honeybee brain, which belongs to the allatostatin‐A (AST) peptide family sharing the conserved c‐terminal sequence ‐YXFGL‐NH2. We found about 500 AST‐immunoreactive (ASTir) neurons in the brain, scattered in 18 groups that varied in their precise location across individuals. Almost all areas of the brain were innervated by ASTir fibers. Most ASTir neurites formed networks within functionally distinct areas, e.g., the antennal lobes, the mushroom bodies, or the optic lobes, indicating local functions of the peptide. A small number of very large neurons had widespread arborizations and neurites were found in the corpora cardiaca and in the cervical connectives, suggesting that AST also has global functions. We double‐stained AST and GABA and found that a subset of ASTir neurons were GABA‐immunoreactive (GABAir). Double staining AST with backfills of olfactory receptor neurons or mass fills of neurons in the antennal lobes and in the mushroom bodies allowed a more fine‐grained description of ASTir networks. Together, this first comprehensive description of AST in the bee brain suggests a diverse functional role of AST, including local and global computational tasks. J. Comp. Neurol. 518:1391–1417, 2010. © 2010 Wiley‐Liss, Inc.

New insights on the neuropeptide Y system in the larval lamprey brain: neuropeptide Y immunoreactive neurons, descending spinal projections and comparison with tyrosine hydroxylase and GABA immunoreactivities

Lampreys are useful models for studying the evolution of the nervous system of vertebrates. Here we used immunofluorescence and tract-tracing methods to study new aspects of the neuropeptide Y-immunoreactive (NPY-ir) system in larval sea lampreys. NPY-ir neurons were observed in brain nuclei that contain NPY-ir cells in other lamprey species. Moreover, a group of NPY-ir cells that migrated away the periventricular layer was observed in the lateral part of the dorsal hypothalamus, which suggests a role for NPY in feeding behavior in lampreys. We also report NPY-ir cells in the dorsal column nucleus, which appears to be unique among vertebrates, and in the habenula. A combination of tract-tracing and immunohistochemical labeling demonstrated the presence of spinal projecting NPY-ir reticular cells in the anterior rhombencephalic reticular formation, and the relationships between the NPY-ir system and the reticulospinal nuclei and some afferent systems. The colocalization of catecholamines and GABA in lamprey NPY-ir neurons was investigated by double immunofluorescence methods. Colocalization of tyrosine hydroxylase (TH) and NPY immunoreactivities was not observed in any brain neuron, although reported in amphibians and mammals. The frequent presence of NPY-ir terminals on TH-ir cells suggests that NPY modulates the activity of some dopaminergic nuclei in lampreys. Colocalization of NPY and GABA immunoreactivities was frequently observed in neurons of different rhombencephalic and diencephalic NPY-ir populations. These results in lampreys suggest that the coexpression of NPY and GABA in neurons appeared early on in the brains of vertebrates.

Differential localization of γ‐aminobutyric acid type a and glycine receptor subunits and gephyrin in the human pons, medulla oblongata and uppermost cervical segment of the spinal cord: An immunohistochemical study

Gephyrin is a multifunctional protein responsible for the clustering of glycine receptors (GlyR) and gamma-aminobutyric acid type A receptors (GABA(A)R). GlyR and GABA(A)R are heteropentameric chloride ion channels that facilitate fast-response, inhibitory neurotransmission in the mammalian brain and spinal cord. We investigated the immunohistochemical distribution of gephyrin and the major GABA(A)R and GlyR subunits in the human light microscopically in the rostral and caudal one-thirds of the pons, in the middle and caudal one-thirds of the medulla oblongata, and in the first cervical segment of the spinal cord. The results demonstrate a widespread pattern of immunoreactivity for GlyR and GABA(A)R subunits throughout these regions, including the spinal trigeminal nucleus, abducens nucleus, facial nucleus, pontine reticular formation, dorsal motor nucleus of the vagus nerve, hypoglossal nucleus, lateral cuneate nucleus, and nucleus of the solitary tract. The GABA(A)R alpha(1) and GlyR alpha(1) and beta subunits show high levels of immunoreactivity in these nuclei. The GABA(A)R subunits alpha(2), alpha(3), beta(2,3), and gamma(2) present weaker levels of immunoreactivity. Exceptions are intense levels of GABA(A)R alpha(2) subunit immunoreactivity in the inferior olivary complex and high levels of GABA(A)R alpha(3) subunit immunoreactivity in the locus coeruleus and raphe nuclei. Gephyrin immunoreactivity is highest in the first segment of the cervical spinal cord and hypoglossal nucleus. Our results suggest that a variety of different inhibitory receptor subtypes is responsible for inhibitory functions in the human brainstem and cervical spinal cord and that gephyrin functions as a clustering molecule for major subtypes of these inhibitory neurotransmitter receptors.

Quantitative study of the developmental changes in calcium‐permeable AMPA receptor‐expressing neurons in the rat somatosensory cortex

The distribution of cells expressing calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) in the somatosensory cortex of rats at different developmental stages was studied using a kainate-stimulated Co(2+)-labeling assay in a quantitative manner. The applicability of this assay for identifying CP-AMPAR-expressing cells was first verified using cultured rat cortical neurons by means of fluorescence Ca(2+) imaging and pharmacological tools. Cells positively identified by the Co(2+)-labelinig assay resided primarily in the marginal zone and subplate of young fetuses and became more widely distributed throughout the cortex as the fetus matured. The majority, >80%, of these Co(2+)-positive cells were neurons, exhibiting immunoreactivity with the neuronal marker NeuN. The proportion of neurons that were Co(2+)-positive increased from approximately 25% to approximately 60% as the rat fetus grew into adulthood. In contrast, less than 20% of nonneuronal cells were Co(2+)-positive. Of the Co(2+)-positive neurons, 15%-31% exhibited GABA immunoreactivity and nonpyramidal-shaped cell bodies; these were presumably GABAergic neurons. Most of the remaining non-GABAergic/Co(2+)-positive neurons had pyramidal-shaped cell bodies and were presumably excitatory principle neurons. Around 70% of GABAergic neurons in the cortex were Co(2+)-positive. Furthermore, in the cortex of neonatal rats the Co(2+)-positive neurons were found to be more susceptible to kainate toxicity than the Co(2+)-negative cells. The Co(2+)-positive neurons in the subplate of neonatal rats were more vulnerable to kainate toxicity than their counterparts in the remaining cortical areas. Together, the widespread distribution and distinct susceptibility to excitotoxicity of CP-AMPAR-expressing neurons suggest that they play various important roles in the development and physiology of the rat cerebral cortex.

Upregulated expression of oncomodulin, the beta isoform of parvalbumin, in perikarya and axons in the diencephalon of parvalbumin knockout mice

The calcium-binding proteins parvalbumin, calbindin D-28k, calretinin and calcineurin are present in subsets of GABAergic gigantic calyciform presynaptic terminals of the reticular thalamic nucleus (RTN). Previously it was hypothesized that GABA and calcium-binding proteins including parvalbumin are not only colocalized in the same neuron subpopulation, but that GABA synthesis and parvalbumin expression could be also genetically regulated by a common mechanism. Moreover, parvalbumin expression levels could influence GABA synthesis. For this, we analyzed GABA immunoreactivity in RTN gigantic calyciform presynaptic terminals of parvalbumin–deficient (PV−/−) mice. With respect to GABA immunoreactivity we found no differences compared to wild–type animals. However, using a polyclonal parvalbumin antibody raised against full-length rat muscle parvalbumin on brain sections of PV−/− mice, we observed paradoxical parvalbumin immunoreactivity in partly varicose axons in the diencephalon, mainly in the lamina medullaris externa surrounding the thalamus. A detailed immunohistochemical, biochemical and molecular biological analysis revealed this immunoreactivity to be the result of an upregulation of oncomodulin (OM), the mammalian beta isoform of parvalbumin in PV−/− mice. In addition, OM was present in a sparse subpopulation of neurons in the thalamus and in the dentate gyrus. OM expression has not been observed before in neurons of the mammalian brain; its expression was restricted to outer hair cells in the organ of Corti. Our results indicate that the absence of parvalbumin has no major effect on the GABA-synthesizing system in RTN presynaptic terminals excluding a direct effect of parvalbumin on this regulation. However, a likely homeostatic mechanism is induced resulting in the upregulation of OM in selected axons and neuronal perikarya. Our results warrant further detailed investigations on the putative role of OM in the brain.

Serotonin and GABA are colocalized in restricted groups of neurons in the larval sea lamprey brain: insights into the early evolution of neurotransmitter colocalization in vertebrates

Colocalization of the classic neurotransmitters serotonin (5-HT) and gamma-aminobutyric acid (GABA) (or the enzyme that synthesizes the latter, glutamate decarboxylase) has been reported in a few neurons of the rat raphe magnus-obscurus nuclei. However, there are no data on the presence of neurochemically similar neurons in the brain of non-mammalian vertebrates. Lampreys are the oldest extant vertebrates and may provide important data on the phylogeny of neurochemical systems. The colocalization of 5-HT and GABA in neurons of the sea lamprey brain was studied using antibodies directed against 5-HT and GABA and confocal microscopy. Colocalization of the neurotransmitters was observed in the diencephalon and the isthmus. In the diencephalon, about 87% of the serotonergic cells of the rostral tier of the dorsal thalamus (close to the zona limitans) exhibited GABA immunoreactivity. In addition, occasional cells double-labelled for GABA and 5-HT were observed in the hypothalamic tuberal nucleus and the pretectum. Of the three serotonergic isthmic subgroups already recognized in the sea lamprey isthmus (dorsal, medial and ventral), such double-labelled cells were only observed in the ventral subgroup (about 61% of the serotonergic cells in the ventral subgroup exhibited GABA immunoreactivity). An equivalence between these lamprey isthmic cells and the serotonergic/GABAergic raphe cells of mammals is suggested. Present findings suggest that serotonergic/GABAergic neurons are more extensive in lampreys than in the rat and probably appeared before the separation of agnathans and gnathostomes. Cotransmission by release of 5-HT and GABA by the here-described lamprey brain neurons is proposed.

Ultrastructural characterization of relationship between serotonergic and GABAergic structures in the ventral part of the oral pontine reticular nucleus

The ventral part of the oral pontine reticular nucleus (vRPO) is involved in the generation and maintenance of rapid eye movement (REM) sleep. Both GABAergic and serotonergic neurotransmission have been implicated in the control of the sleep–wakefulness cycle. Nevertheless, the synaptic organization of serotonergic terminals in the vRPO has not yet been characterized. We performed an electron microscope study of serotonin-immunoreactive (5-HT-IR) terminals using immunoperoxidase or immunogold–silver methods. In a second set of experiments, combining GABA immunoperoxidase and 5-HT immunogold–silver techniques, we examined inputs from GABA-immunoreactive (GABA-IR) terminals to serotonergic neurons. 5-HT-IR terminals were located primarily on dendrites and occasionally on somata of unlabeled and 5-HT-IR neurons. The majority of the synapses formed by 5-HT-IR terminals were of the symmetrical type, making contacts primarily with unlabeled dendritic profiles. Moreover, 5-HT-IR terminals contacted unlabeled axon terminals that formed asymmetric synapses on dendrites. Double immunolabeling experiments showed 5-HT-IR and GABA-IR afferents, in apposition to each other, making synapses with the same dendrites. Finally, GABA-IR terminals innervated 5-HT-IR and GABA-IR dendrites. Our findings indicate that serotonin would modulate the neuronal activity through inhibitory or excitatory influences, although the action of serotonin on the vRPO would predominantly be inhibitory. Moreover, the present results suggest that the serotonin modulation of vRPO neurons might involve indirect connections. In addition, GABA might contribute to the induction and maintenance of REM sleep by inhibiting serotonergic and GABAergic neurons in the vRPO.

Involvement of ERK phosphorylation in brainstem neurons in modulation of swallowing reflex in rats

In order to evaluate the neuronal mechanisms underlying functional abnormalities of swallowing in orofacial pain patients, this study investigated the effects of noxious orofacial stimulation on the swallowing reflex, phosphorylated extracellular signal-regulated kinase (pERK) and gamma-aminobutyric acid (GABA) immunohistochemical features in brainstem neurons, and also analysed the effects of brainstem lesioning and of microinjection of GABA receptor agonist or antagonist into the nucleus tractus solitarii (NTS) on the swallowing reflex in anaesthetized rats. The swallowing reflex elicited by topical administration of distilled water to the pharyngolaryngeal region was inhibited after capsaicin injection into the facial (whisker pad) skin or lingual muscle. The capsaicin-induced inhibitory effect on the swallowing reflex was itself depressed after the intrathecal administration of MAPK kinase (MEK) inhibitor. No change in the capsaicin-induced inhibitory effect was observed after trigeminal spinal subnucleus caudalis lesioning, but the inhibitory effect was diminished by paratrigeminal nucleus (Pa5) lesioning. Many pERK-like immunoreactive neurons in the NTS showed GABA immunoreactivity. The local microinjection of the GABA(A) receptor agonist muscimol into the NTS produced a significant reduction in swallowing reflex, and the capsaicin-induced depression of the swallowing reflex was abolished by microinjection of the GABA(A) receptor antagonist bicuculline into the NTS. The present findings suggest that facial skin-NTS, lingual muscle-NTS and lingual muscle-Pa5-NTS pathways are involved in the modulation of swallowing reflex by facial and lingual pain, respectively, and that the activation of GABAergic NTS neurons is involved in the inhibition of the swallowing reflex following noxious stimulation of facial and intraoral structures.

γ-Aminobutyric acid immunostaining in the antennal lobe of the moth Heliothis virescens and its colocalization with neuropeptides

The antennal lobe is the primary processing center for olfactory information in insects. To understand further the neural circuitry of this brain area, we have investigated the distribution of gamma-aminobutyric acid (GABA) and its colocalization with neuropeptides in the antennal lobe of the noctuid moth Heliothis virescens. Immunocytochemical experiments with an antiserum against GABA showed a large number of labeled somata in the antennal lobe; these somata were located exclusively in the lateral cell cluster. Stained neurites innervating all antennal-lobe glomeruli, including the male-specific macroglomerular complex, suggested a prominent role of GABA in processing olfactory information, including signals from pheromones, interspecifically acting odors, and plant odors. Fibers in two antennocerebral tracts (the middle and dorsal antennocerebral tract) exhibited prominent GABA immunoreactivity. Double-labeling experiments revealed that immunostaining for three neuropeptides, viz., A-type allatostatin, Manduca sexta allatotropin, and FMRFamide-related peptides, was largely colocalized with GABA in cell bodies of the lateral cell cluster. The general absence of peptide immunostaining in the antennocerebral tracts strongly indicated that these peptides were colocalized with GABA in local interneurons of the antennal lobe. In contrast, tachykinin-related peptides occurred in a distinct population of local antennal-lobe neurons that did not exhibit GABA immunostaining. Thus, local interneurons that were not GABAergic were present in the moth antennal lobe.

Neurokinin-1 Receptor Immunoreactive Neuronal Elements in the Superficial Dorsal Horn of the Chicken Spinal Cord: With Special Reference to Their Relationship with the Tachykinin-containing Central Axon Terminals in Synaptic Glomeruli

Synaptic glomeruli that involve tachykinin-containing primary afferent central terminals are numerous in lamina II of the chicken spinal cord. Therefore, a certain amount of noxious information is likely to be modulated in these structures in chickens. In this study, we used immunohistochemistry with confocal and electron microscopy to investigate whether neurokinin-1 receptor (NK-1R)-expressing neuronal elements are in contact with the central primary afferent terminals in synaptic glomeruli of the chicken spinal cord. We also investigated which neuronal elements (axon terminals, dendrites, cell bodies) and which neurons in the spinal cord possess NK-1R, and are possibly influenced by tachykinin in the glomeruli. By confocal microscopy, NK-1R immunoreactivities were seen in a variety of neuronal cell bodies, their dendrites and smaller fibers of unknown origin. Some of the NK-1R immunoreactive profiles also expressed GABA immunoreactivities. A close association was observed between the NK-1R-immunoreactive neurons and tachykinin-immunoreactive axonal varicosities. By electron microscopy, NK-1R immunoreactivity was seen in cell bodies, conventional dendrites and vesicle-containing dendrites in laminae I and II. Among these elements, dendrites and vesicle-containing dendrites made contact with tachykinin-containing central terminals in the synaptic glomeruli. These results indicate that tachykinin-containing central terminals in the chicken spinal cord can modulate second-order neuronal elements in the synaptic glomeruli.

Differential Expression of GABA Receptor Subunit Messenger Ribonucleic Acids and Immunoreactivity in the Rat Neostriatum during Postnatal Development

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the neostriatum. Functions of GABA are mediated by GABA_A and GABA_B receptors in the neostriatum. In order to investigate the developmental expression of GABA receptor subunits (GABA_Aα₁, GABA_Aα₃ and GABA_Aα₆; GABA_Aβ₂ and GABA_Aβ₃, and GABA_BR₁ and GABA_BR₂) in the rat neostriatum, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence were performed. Tissues were obtained from rats on postnatal day (PND) 1, PND 7 and PND 14, and from adult rats. RT-PCR indicated that GABA_Aα₁ and GABA_Aα₆ mRNA levels were low, but that GABA_Aβ₂ and GABA_Aβ₃ mRNAs levels were high during the early postnatal period. Immunofluorescence revealed that GABA_Aα₁ immunoreactivity was only observed in striatal interneurons in PND 14 and in adult rats. Immunoreactivity for GABA_Aα₆ was only observed in PND 14 and adult animals. GABA_BR₁ immunoreactivity was found to be expressed by perikarya of striatal neurons at all ages examined. In contrast, GABA_BR₂ immunoreactivity was mainly observed in choline acetyltransferase-positive striatal interneurons in PND 14 and adult rats. The present results indicate that there are differential patterns of developmental expression of GABA receptor subunits in the neostriatum, with important implications for the development of GABA systems in rat basal ganglia.

Ultrastructural analysis of glutamate‐, GABA‐, and glycine‐immunopositive boutons from supratrigeminal premotoneurons in the rat trigeminal motor nucleus

The supratrigeminal region (Vsup) is important for coordination of smooth jaw movement. However, little is known about the synaptic connections of the Vsup premotoneurons with the trigeminal motor neurons. In the present study, we examined axon terminals of Vsup premotoneurons in the contralateral trigeminal motor nucleus (Vmo) by a combination of anterograde tracing with cholera toxin B-horseradish peroxidase (CTB-HRP), postembedding immunohistochemistry for the amino acid transmitters glutamate, GABA, and glycine, and electron microscopy. Tracer injections resulted in anterograde labeling of axon terminals of the Vsup premotoneurons in the motor trigeminal nucleus (Vmo). The labeled boutons in Vmo exhibited immunoreactivity for glutamate, GABA, or glycine: glutamate-immunopositive boutons (69%) were more frequently observed than GABA- or glycine-immunopositive boutons (19% and 12%, respectively). Although most labeled boutons (97%) made synaptic contacts with a single postsynaptic dendrite, a few glutamate-immunopositive boutons (3%) showed synaptic contact with two dendrites. No labeled boutons participated in axoaxonic synaptic contacts. Most labeled boutons (78%) were presynaptic to dendritic shafts, and the remaining 22% were presynaptic to somata or primary dendrites. A large proportion of GABA- or glycine-immunopositive boutons (40%) were presynaptic to somata or primary dendrites, whereas most glutamate-immunopositive boutons (86%) were presynaptic to dendritic shafts. These results indicate that axon terminals of Vsup premotoneurons show simple synaptic connection with Vmo neurons. This may provide the anatomical basis for the neural information processing responsible for jaw movement control.

GABA is not elevated during neuroprotective neuronal depression in the hypoxic epaulette shark ( Hemiscyllium ocellatum)

Prolonged hypoxic exposure results in cell failure, glutamate excitotoxicity and apoptosis in the brain. The epaulette shark can withstand prolonged hypoxic exposure without brain injury, while maintaining normal function and activity at tropical temperatures. We examined whether the inhibitory neurotransmitter GABA was involved in hypoxia tolerance and neuroprotection during hypoxic preconditioning. Sharks were exposed to either cyclic hypoxic preconditioning or normoxic conditions. Whole brain GABA concentration was determined using high performance liquid chromatography; GABA distribution in neuronal structures was localised with immunohistochemistry and quantified. While the overall brain level of GABA was not significantly different, there was a significant heterogeneous change in GABA distribution. GABA immunoreactivity was elevated in key motor and sensory nuclei from preconditioned animals, including the nucleus motorius nervi vagi and the cerebellar crest ( p < 0.001), corresponding to areas of previously reported neuronal hypometabolism. Since the neuroprotection in all other hypoxia and anoxia tolerant species examined so far relies in part on significant elevations in GABA and the phylogenetically older epaulette shark does not, it is reasonable to assume that further research in this unique animal model may yield clues to new key modulators of neuroprotection. Understanding such mechanisms may facilitate the development of therapeutic interventions in the treatment of transient ischaemic attacks, strokes and traumatic brain injury.

Neurochemical characterization of sea lamprey taste buds and afferent gustatory fibers: Presence of serotonin, calretinin, and CGRP immunoreactivity in taste bud bi‐ciliated cells of the earliest vertebrates

Neuroactive substances such as serotonin and other monoamines have been suggested to be involved in the transmission of gustatory signals from taste bud cells to afferent fibers. Lampreys are the earliest vertebrates that possess taste buds, although these differ in structure from taste buds in jawed vertebrates, and their neurochemistry remains unknown. We used immunofluorescence methods with antibodies raised against serotonin, tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA), glutamate, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), calretinin, and acetylated alpha-tubulin to characterize the neurochemistry and innervation of taste buds in the sea lamprey, Petromyzon marinus L. For localization of proliferative cells in taste buds we used bromodeoxyuridine labeling and proliferating cell nuclear antigen immunohistochemistry. Results with both markers indicate that proliferating cells are restricted to a few basal cells and that almost all cells in taste buds are nonproliferating. A large number of serotonin-, calretinin-, and CGRP-immunoreactive bi-ciliated cells were revealed in lamprey taste buds. This suggests that serotonin participates in the transmission of gustatory signals and indicates that this substance appeared early on in vertebrate evolution. The basal surface of the bi-ciliated taste bud cells was contacted by tubulin-immunoreactive fibers. Some of the fibers surrounding the taste bud were calretinin immunoreactive. Lamprey taste bud cells or afferent fibers did not exhibit TH, GABA, glutamate, or NPY immunoreactivity, which suggests that expression of these substances evolved in taste buds of some gnathostomes lines after the separation of gnathostomes and lampreys.

Some γ-motoneurons contain γ-aminobutyric acid in the rat cervical spinal cord

γ-aminobutyric acid (GABA) is utilized in the peripheral as well as central nervous system. In this study, fibers immunoreactive for 67 kDa isoform of glutamic acid decarboxylase (GAD67), an enzyme which synthesizes GABA, were found to terminate in the intercapsular region of muscle spindles of the upper limb. GABA-containing fibers were also found in the ventral roots of C5 to T5 spinal segments, brachial plexus, and radial nerve. These fibers were thin and immunoreactive for choline-acetyl transferase (ChAT). After transection of the brachial plexus, GABA immunoreactivity disappeared completely in the ipsilateral triceps brachii muscle (TBM). After the injection of fluorogold into the TBM, some retrogradely labeled medium-sized neurons were positive for GAD67, but not VGAT mRNA. All these observations clearly indicate that GABA-containing γ-motoneurons in the lower cervical spinal cord send their fibers to muscle spindles in the upper extremities. Since we detected neither GABA A nor GABA B receptors in the TBM by RT-PCR, the function of the GABA-containing γ-motoneurons remains unclear.

NbActiv4 medium improvement to Neurobasal/B27 increases neuron synapse densities and network spike rates on multielectrode arrays

The most interesting property of neurons is their long-distance propagation of signals as spiking action potentials. Since 1993, Neurobasal/B27™ has been used as a serum-free medium optimized for hippocampal neuron survival. Neurons on microelectrode arrays (MEA) were used as an assay system to increase spontaneous spike rates in media of different compositions. We find spike rates of 0.5 s −1 (Hz) for rat embryonic hippocampal neurons cultured in Neurobasal/B27, lower than cultures in serum-based media and offering an opportunity for improvement. NbActiv4™ was formulated by addition of creatine, cholesterol and estrogen to Neurobasal/B27 that synergistically produced an eightfold increase in spontaneous spike activity. The increased activity with NbActiv4 correlated with a twofold increase in immunoreactive synaptophysin bright puncta and GluR1 total puncta. Characteristic of synaptic scaling, immunoreactive GABA Aβ puncta also increased 1.5-fold and NMDA-R1 puncta increased 1.8-fold. Neuron survival in NbActiv4 equaled that in Neurobasal/B27, but with slightly higher astroglia. Resting respiratory demand was decreased and demand capacity was increased in NbActiv4, indicating less stress and higher efficiency. These results show that NbActiv4 is an improvement to Neurobasal/B27 for cultured networks with an increased density of synapses and transmitter receptors which produces higher spontaneous spike rates in neuron networks.

Distribution of glycine immunoreactivity in the brain of adult sea lamprey (Petromyzon marinus). Comparison with γ‐aminobutyric acid

The distribution of glycinergic cells in the brain of nonmammalian vertebrates is still unknown. Lampreys are the most primitive extant vertebrates, and they may provide important data on the phylogeny of this system. Here, we studied for the first time the distribution of glycine immunoreactivity in the sea lamprey brain and compared it with gamma-aminobutyric acid (GABA)-ergic populations. Most glycine-immunoreactive neurons were found at midbrain and hindbrain levels, and most of these cells did not exhibit GABA immunoreactivity. We describe glycine-immunoreactive cell populations in the olfactory bulbs, the preoptic nucleus, and the thalamus of the sea lamprey, which is in striking contrast to their lack in the mammalian forebrain. We also observed glycine-immunoreactive populations in the optic tectum, the torus semicircularis and the midbrain tegmentum, the isthmus, the octavolateral area, the dorsal column nucleus, the abducens nucleus, the trigeminal motor nucleus, the facial motor nucleus, and the rhombencephalic reticular formation. In these populations, colocalization with GABA was observed in only some cells of the tegmental M5 nucleus, ventral isthmus, medial octavolateral nucleus, dorsal column nucleus, and lateral reticular region. The present results allow us to conclude that the distribution of glycine-immunoreactive cells changed notably from lamprey to mammals, with a decrease in glycinergic populations in the forebrain and a specialization of brainstem cell groups. Although knowledge of the glycinergic populations in lampreys is important for understanding the early evolution of this system, there is a notable gap of information regarding its organization in brains of other nonmammalian vertebrates.

Neurochemical differentiation of horizontal and amacrine cells during transformation of the sea lamprey retina

The sea lamprey is a modern representative of the earliest vertebrates (the agnathans) in which development of the eye and retina shows unique patterns. In larval stages the retina is poorly developed, and although a small central region has developed glutamatergic vertical pathways, there is no evidence of chemical differentiation of amacrine and horizontal cells in the central or lateral larval retina [Villar-Cerviño, V., Abalo, X.M., Villar-Cheda, B., Meléndez-Ferro, M., Pérez-Costas, E., Holstein, G.R., Martinelli, G.P., Rodicio, M.C., Anadón, R., 2006. Presence of glutamate, glycine, and γ-aminobutyric acid in the retina of the larval sea lamprey: comparative immunohistochemical study of classical neurotransmitters in larval and postmetamorphic retinas. J. Comp. Neurol. 499, 810–827.]. However, in adults all the retina was differentiated and both amacrine and horizontal cells are well developed. Present immunocytochemical results show that the horizontal and amacrine cells of the retina begin their neurochemical differentiation during metamorphosis, when they start to express GABA, glycine, serotonin and dopamine; this occurs several years after the onset of development. Immunoreactivity for GABA, glycine and serotonin was found at early metamorphic stages, while expression of the markers of catecholaminergic amacrine cells, dopamine and tyrosine hydroxylase, was found to be delayed until intermediate metamorphic stages. GABA, which is found in some amacrine and horizontal cells of adults, was first observed in amacrine cells during early stages of transformation and then in horizontal cells during middle stages. All cells immunoreactive to serotonin or tyrosine hydroxylase/dopamine were amacrine cells. Interestingly, all these markers began expression before the appearance of opsin-immunoreactive photoreceptors in the lateral retina. The pattern of chemical differentiation of amacrine and horizontal cells was compared with that of other vertebrates and their significance was discussed.