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PubMed1998

Voltage-activated currents in identified giant interneurons isolated from adult crickets gryllus bimaculatus

The electrophysiological properties of cultured giant interneurons isolated from the terminal ganglion of adult crickets (Gryllus bimaculatus) were investigated using whole-cell patch-clamp techniques. To allow for unequivocal identification of these interneurons in cell culture, a protocol for fast and selective labeling of their cell bodies was established. Prior to cell dissociation, the giant interneurons were backfilled through their axons in situ with a fluorescent dye (dextran tetramethylrhodamine). In primary cell cultures, the cell bodies of giant interneurons were identified among a population of co-cultured neurons by their red fluorescence. Action potentials were recorded from the cell bodies of the cultured interneurons suggesting that several types of voltage-activated ion channels exist in these cells. Using voltage-clamp recording techniques, four voltage-activated currents were isolated and characterized. The giant interneurons express at least two distinct K+ currents: a transient current that is blocked by 4-aminopyridine (4x10(-3 )mol l-1) and a sustained current that is partially blocked by tetraethylammonium (3x10(-2 )mol l-1) and quinidine (2x10(-4 )mol l-1). In addition, a transient Na+ current sensitive to 10(-7 )mol l-1 tetrodotoxin and a Ca2+ current blocked by 5x10(-4 )mol l-1 CdCl2 have been characterized. This study represents the first step in an attempt to analyze the cellular and ionic mechanisms underlying plasticity in the well-characterized and behaviorally important giant interneuron pathway in insects.

Journal Article↗
PubMed1991

Deficits in small interneurons in prefrontal and cingulate cortices of schizophrenic and schizoaffective patients.

A recent report suggested that neurons in the prefrontal, anterior cingulate, and primary motor cortex of the brains of schizophrenic subjects may be less dense than those in the brains of nonschizophrenic subjects. We have determined whether pyramidal neurons and/or interneurons are preferentially reduced in schizophrenic subjects. Twelve control subjects and 18 schizophrenic subjects were studied in a blind, quantitative analysis of the density of pyramidal cells, interneurons, and glial cells in each of the six layers of the anterior cingulate and prefrontal cortex. The results showed that numbers of small neurons (interneurons) were reduced in most layers of the cingulate cortex in schizophrenic subjects compared with nonschizophrenic subjects, with the differences being greatest in layer II. In the prefrontal area, interneuronal density was also lower in layer II and, to a lesser extent, in layer I in schizophrenic subjects compared with control subjects. In most cases, the differences were similar, although more significant, in schizophrenic subjects who had had superimposed mood disturbances than in schizophrenic subjects who had not had such comorbidity. Numbers of pyramidal neurons generally were not different between control and schizophrenic subjects, except in layer V of the prefrontal area, where schizophrenic subjects showed higher densities of these neurons. Glial numbers did not differ between the control and schizophrenic subjects, suggesting that a neurodegenerative process did not cause the reduced interneuronal density observed. Using multiple regression analysis and analysis of covariance, decreases in the density of layer II interneurons could not be adequately explained by the effects of various confounding variables, such as age, postmortem interval, duration of specimen fixation, or administration of neuroleptic agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Count↗
PubMed1997

Localization of last-order premotor interneurons in the lumbar spinal cord of rats.

There is strong evidence that neural circuits underlying certain rhythmic motor behaviors are located in the spinal cord. Such local central pattern generators are thought to coordinate the activity of motoneurons through specific sets of last-order premotor interneurons that establish monosynaptic contacts with motoneurons. After injections of biotinylated dextran amine into the lateral and medial motor columns as well as the ventrolateral white matter at the level of the upper and lower segments of the lumbar spinal cord, we intended to identify and localize retrogradely labelled spinal interneurons that can likely be regarded as last-order premotor interneurons in rats. Regardless of the location of the injection site, labelled interneurons were revealed in laminae V-VIII along a three- or four-segment-long section of the spinal gray matter. Although most of the stained cells were confined to laminae V-VIII in all cases, the distribution of neurons within the confines of this area varied according to the site of injection. After injections into the lateral motor column at the level of the L4-L5 segments, the labelled neurons were located almost exclusively in laminae V-VII ipsilateral to the injection site, and the perikarya were distributed throughout the entire mediolateral extent of this area. Interneurons projecting to the lateral motor column at the level of the L1-L2 segments were also located in laminae V-VII, but most of them were concentrated in the middle one-third or in the lateral half of this area. Following injections into the medial motor column at the level of the L1-L2 segments, the majority of labelled neurons were confined to the medial aspect of laminae V-VII and lamina VIII, and the proportion of neurons that were found contralateral to the injection site was strikingly higher than in the other experimental groups. The results suggest that the organization of last-order premotor interneurons projecting to motoneurons, which are located at different areas of the lateral and medial motor columns and innervate different muscle groups, may present distinct features in the rat spinal cord.

Animals↗
PubMed2000

Spike-dependent calcium influx in dendrites of the cricket giant interneuron.

Identified wind-sensitive giant interneurons in the cricket's cercal sensory system integrate cercal afferent signals and release an avoidance behavior. A calcium-imaging technique was applied to the giant interneurons to examine the presence of the voltage-dependent Ca(2+) channels (VDCCs) in their dendrites. We found that presynaptic stimuli to the cercal sensory nerve cords elevated the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in the dendrites of the giant interneurons. The dendritic Ca(2+) rise coincided with the spike burst of the giant interneurons, and the rate of Ca(2+) rise depended on the frequency of the action potentials. These results suggest that the action potentials directly caused [Ca(2+)](i) increase. Observation of the [Ca(2+)](i) elevation induced by depolarizing current injection demonstrates the presence of the VDCCs in the dendrites. Although hyperpolarizing current injection into the giant interneuron suppressed action potential generation, EPSPs could induce no [Ca(2+)](i) increase. This result means that ligand-gated channels do not contribute to the synaptically stimulated Ca(2+) elevation. On the other hand, antidromically stimulated spikes also increased [Ca(2+)](i) in all cellular regions including the dendrites. And bath application of a mixture of Ni(2+), Co(2+), and Cd(2+) or tetrodotoxin inhibited the [Ca(2+)](i) elevation induced by the antidromic stimulation. From these findings, we suppose that the axonal spikes antidromically propagate and induce the Ca(2+) influx via VDCCs in the dendrites. The spike-dependent Ca(2+) elevation may regulate the sensory signals processing via second-messenger cascades in the giant interneurons.

Action Potentials↗
PubMed2002

Axon arbors and synaptic connections of a vulnerable population of interneurons in the dentate gyrus in vivo.

The predominant gamma-aminobutyric acid (GABA)ergic neuron class in the hilus of the dentate gyrus consists of spiny somatostatinergic interneurons. We examined the axon projections and synaptic connections made by spiny hilar interneurons labeled with biocytin in gerbils in vivo. Axon length was 152-497 mm/neuron. Sixty to 85% of the axon concentrated in the outer two thirds of the molecular layer of the dentate gyrus. The septotemporal span of the axon arbor extended over 48-82% of the total hippocampal length, which far exceeds the septotemporal span of axons of granule cells whose complete axon arbors extended over 15-29%. A three-dimensionally reconstructed 216-microm-long spiny hilar interneuron axon segment in the outer third of the molecular layer formed an average of 1 synapse every 5.1 microm. Of the 42 symmetric (inhibitory) synapses formed by the reconstructed segment, 88% were with spiny dendrites of presumed granule cells, and 67% were with dendritic spines that also receive an asymmetric (excitatory) contact from an unlabeled axon terminal. Postembedding GABA-immunocytochemistry revealed that 55% of the GABAergic synapses in the outer third of the molecular layer were with spines. Therefore, in the outer molecular layer, spiny hilar interneurons form synaptic contacts that appear to be positioned to exert inhibitory control near sites of excitatory synaptic input from the entorhinal cortex to granule cell dendritic spines. These findings demonstrate far-reaching, yet highly specific, connectivity of individual interneurons and suggest that the loss of spiny hilar interneurons, as occurs in temporal lobe epilepsy, may contribute to hyperexcitability in the hippocampus.

Action Potentials↗
PubMed2004

Neurotransmitter properties of spinal interneurons in embryonic and larval zebrafish.

Many classes of spinal interneurons in zebrafish have been described based on morphology, but their transmitter phenotypes are largely unknown. Here we combine back-filling or genetic labeling of spinal interneurons with in situ staining for markers of neurotransmitter phenotypes, including the vesicular glutamate transporter (VGLUT) genes for glutamatergic neurons, the neuronal glycine transporter (GLYT2) for glycinergic neurons, and glutamic acid decarboxylase (GAD) for GABAergic neurons. Neurons positive for VGLUT include the commissural CoPA, MCoD, UCoD, and some of the CoSA neurons. The CiD interneurons, which have ipsilateral descending axons, were also VGLUT-positive, as were the ventrally located VeMe interneurons, whose descending axonal trajectory has not been clearly revealed. Cells positive for GLYT2 include the commissural CoLAs as well as some of the CoBL and CoSA neurons. The CiA cells were the only GLYT2-positive cells with an ipsilateral axon. Cells staining for GAD included, most notably, the dorsal longitudinal ascending (DoLA) and KA interneurons. Our approach allowed us to define the likely transmitter phenotypes of most of the known classes of spinal interneurons. These data provide a foundation for understanding the functional organization of the spinal networks in zebrafish.

Amino Acid Transport Systems, Neutral↗
PubMed2007

Heterogeneity of glycinergic and gabaergic interneurons in the granule cell layer of mouse cerebellum.

Interneurons of the cerebellum granule cell layer (GCL) form distinct populations. Golgi cells extend dendrites in the molecular layer (ML) and innervate granule cells. In contrast, Lugaro cells have dendrites confined to the GCL but innervate interneurons in the ML, and globular cells have both their dendrites and axons in the ML. The latter cells were described recently and remain poorly characterized. Although several neurochemical markers have been associated selectively with GCL interneurons, it is unclear how they relate to their morphological classification and neurochemical phenotype (glycinergic and/or gamma-aminobutyric acid [GABA]ergic). Here, we performed a detailed characterization of GCL interneurons in mice expressing enhanced green fluorescent protein (GFP) in glycinergic and GABAergic neurons, respectively. By using immunofluorescence for metabotropic glutamate receptor 2 (mGluR2) and neurogranin as markers, we demonstrate the existence of five non-overlapping subsets of Golgi cells: about 65% are glycinergic/GABAergic and co-express both markers. Two small subsets (5-10%) also contain both neurotransmitters but express only mGluR2; they are distinguished by cell body size and location in the GCL. The fourth subset (15%) is GABAergic only and expresses neurogranin. The fifth subset (5%) is glycinergic only and lacks both markers. Thus, the heterogeneity of Golgi cells suggests that they belong to specific functional circuits and are differentially regulated by mGluRs and Ca(2+)-calmodulin-dependent signaling pathways. In contrast to Golgi cells, Lugaro and globular cells are glycinergic/GABAergic and lack mGluR2 and neurogranin. They each represent at least 15% of GCL interneurons and extensively innervate stellate and basket cells, but not Purkinje cells, emphasizing their contribution to inhibitory control of ML interneurons.

Animals↗
PubMed2007

Selective loss of dentate hilar interneurons contributes to reduced synaptic inhibition of granule cells in an electrical stimulation-based animal model of temporal lobe epilepsy.

Neuropeptide-containing hippocampal interneurons and dentate granule cell inhibition were investigated at different periods following electrical stimulation-induced, self-sustaining status epilepticus (SE) in rats. Immunohistochemistry for somatostatin (SOM), neuropeptide Y (NPY), parvalbumin (PV), cholecystokinin (CCK), and Fluoro-Jade B was performed on sections from hippocampus contralateral to the stimulated side and studied by confocal laser scanning microscopy. Compared to paired age-matched control animals, there were fewer SOM and NPY-immunoreactive (IR) interneurons in the hilus of the dentate gyrus in animals with epilepsy (40-60 days after SE), and 1, 3, and 7 days following SE. In the hilus of animals that had recently undergone SE, some SOM-IR and NPY-IR interneurons also stained for Fluoro-Jade B. Furthermore, there was electron microscopic evidence of the degeneration of SOM-IR interneurons following SE. In contrast, the number of CCK and PV-IR basket cells in epileptic animals was similar to that in controls, although it was transiently diminished following SE; there was no evidence of degeneration of CCK or PV-IR interneurons. Patch-clamp recordings revealed a diminished frequency of inhibitory postsynaptic currents in dentate granule cells (DGCs) recorded from epileptic animals and animals that had recently undergone SE compared with controls. These results confirm the selective vulnerability of a particular subset of dentate hilar interneurons after prolonged SE. This loss may contribute to the reduced GABAergic synaptic inhibition of granule cells in epileptic animals.

Animals↗
PubMed1986

Distribution and ultrastructure of synapses on a premotor local nonspiking interneuron of the crayfish.

Many premotor local nonspiking interneurons are involved in the control of the uropod movements of a crayfish. One of these interneurons was impaled intracellularly, characterized physiologically, and then labeled by intracellular horseradish peroxidase (HRP) injection to examine the distribution and ultrastructure of synapses. Depolarization of this interneuron by a current injection excited the closer motoneurons of the uropod and inhibited its opener motoneurons, but hyperpolarization had no effect. Input and output synapses are distributed all over the major branches and the finer neurites except for the main neurite (7-10 micron in diameter), which runs near the dorsal surface of the neuropil. Both types of synapse are located on the same neurite and are often intermingled in close proximity, often less than 1 micron apart. Presynaptic terminals of the interneuron contain round, clear vesicles that are densely packed in fine branches and spines. The number of synaptic vesicles associated with a particular output synapse was estimated to be about 2,000. No dense-cored granules are observed in the labeled neurites. Our results support the proposal that synaptic transmission in this class of interneurons of the crayfish can sometimes be restricted to a very small region of the branches and that, therefore, different regions of the interneuron can function independently.

Animals↗
PubMed1989

Morphology of a new population of spiking local interneurones in the locust metathoracic ganglion.

A new population of spiking local interneurones is described in the metathoracic ganglion of the locust. All have their cell bodies in a cluster at the ventral, anteromedial portion of the ganglion close to the inner edge of an anterior connective. Their branches are restricted to the same half of the ganglion as their cell bodies. Fifteen types of these unilateral interneurones have been characterized by their morphology, as revealed by intracellular injection of dye, and by their physiology, as revealed by intracellular recording. Each interneurone has a characteristic shape and physiological action, but they form three distinct subgroups according to their gross morphology and their mode of operation. Interneurones of the first subgroup have ventral branches that extend over most of one-half of the metathoracic neuropil and receive inputs mainly from exteroceptors on the ipsilateral hind leg. Typically, they have extensive and reciprocal receptive fields along the dorsoventral axis of the hind leg. Interneurones of the second subgroup have two main ventral branches which extend either medially or laterally and receive inputs mainly from proprioceptors of the hind leg. Interneurones of the third subgroup have few ventral branches and are inhibited by inputs anywhere on the hind leg.

Action Potentials↗
PubMed1989

Nonspiking interneurons in the ventilatory central pattern generator of the shore crab, Carcinus maenas.

Eight nonspiking interneurons were identified that are elements of the central pattern generator controlling ventilation in the shore crab, Carcinus maenas. Intracellular recordings from these neurons in an isolated ganglion preparation revealed that these cells exhibit large amplitude oscillations in their membrane potentials, which are in-phase with the ventilatory motor pattern. These oscillations are present during the expression of the two distinct ventilatory motor output patterns corresponding to forward and reversed ventilation, and the oscillations stopped during pauses in the ventilatory rhythm. Injection of intracellular current pulses into these interneurons caused a resetting of the ongoing ventilatory rhythm, indicating that these cells are part of the ventilatory central pattern generator. The structure of each interneuron was determined by the intracellular injection of Lucifer Yellow dye. These neurons have a large diameter main neurite ranging from 10 to 20 microns in diameter with very restricted primary and secondary branching from the main neurite. All of the interneurons are restricted to a single hemiganglion and perturbation of these cells with intracellular current pulses only affect the motor output of the hemiganglion containing the interneuron. These eight nonspiking interneurons appear to be the primary components of the central pattern generator underlying ventilation in the crab.

Animals↗
PubMed1990

GABA-like immunoreactivity in a population of locust intersegmental interneurones and their inputs.

Intracellular labelling of locust intersegmental interneurones with lucifer yellow or horseradish peroxidase was carried out in combination with light and electron microscope immunocytochemistry by using an antibody raised against gamma amino butyric acid (GABA). Fifteen percent (four out of 27) of intracellularly stained interneurones showed GABA-like immunoreactivity. This is in agreement with previous physiological observations that 20% of the interneurones in this population make inhibitory output connections in the metathoracic ganglion. GABA-like immunoreactivity was also found in processes presynaptic to the interneurones in the mesothoracic ganglion. The presence of such immunoreactive inputs onto the intersegmental interneurones correlates well with physiological evidence that their receptive fields are in part shaped by direct input from GABA-ergic spiking local interneurones.

Afferent Pathways↗
PubMed1991

Anatomy and physiology of spiking local and intersegmental interneurons in the median neuroblast lineage of the grasshopper.

The range of anatomical and physiological properties in the adult progeny of an identified neuroblast was investigated. Some 80-90 adult neurons constitute the dorsal unpaired median (DUM) group of the grasshopper metathoracic ganglion. Within the group are efferent, octopaminergic neurons with large cell bodies and overshooting action potentials. Our objective was to determine the properties of the neurons with small cell bodies that make up the majority of the clone, some 60-70 neurons, about which scant information was available. The small DUM neurons have cell body diameters of 10-20 microns and stain with antibodies to GABA (Thompson and Siegler, '89: Proc. Soc. Neurosci. 15:1296 (abstr.); Witten and Truman, '89: Proc. Soc. Neurosci. 15:365 (abstr.)). By employing intracellular electrophysiological and morphological techniques, we have established that the small DUM neurons are spiking interneurons, expressing passively conducted action potentials in the cell body. They fall into two basic classes: local interneurons with bilateral branches in the auditory neuropiles, and intersegmental interneurons with bilateral branches widespread in the methathoracic ganglion and axons traveling in both anterior connectives. The local interneurons typically respond to sound, whereas the intersegmental interneurons selectively respond to wind on the head or to generalized movements by the animal. Primary neurites of small and large DUM neurons enter the neuropil in a bundle, but the neurites of DUM interneurons are more posterior and have a separate trajectory from those of the efferent DUM neurons once in the ganglion core. A model is presented for the sequential development of efferent, local, and intersegmental DUM neurons from the median neuroblast.

Action Potentials↗
PubMed1993

Distribution of GABAergic synaptic terminals on the dendrites of locust spiking local interneurones.

Double-labelling and electron microscopy were used to assess the distribution of GABAergic synapses made onto the neurites of spiking local interneurones in the locust. The aims were to determine the sites of inputs mediating inhibition of the spiking local interneurones and to ascertain the relative abundance of such inputs. This information should allow us to understand better the integrative properties of these spiking local interneurones and the role of inhibition in shaping their receptive field properties or in fine tuning their spike-mediated outputs. Spiking interneurones in a midline population were labelled by intracellular injection of horseradish peroxidase after physiological characterisation. Colloidal gold immunocytochemistry was then used on ultrathin sections of these neurones with a polyclonal antibody raised against GABA. Most GABAergic (inhibitory) input synapses onto the interneurones are made on their ventral neurites, which also receive afferent (excitatory) inputs. These inhibitory inputs to the ventral neurites constitute 43% of the identifiable synapses. Relatively few GABAergic inputs were found onto the dorsal neurites, which are predominantly the sites of output synapses from these interneurones. These results suggest that much synaptic integration takes place in the ventral field of branches and that GABA-mediated presynaptic inhibitory control of spike-mediated outputs from the dorsal neurites is unlikely to occur.

3,3'-Diaminobenzidine↗
PubMed1993

Morphology of interneurones in pathways from group II muscle afferents in sacral segments of the cat spinal cord.

The morphology of 12 sacral interneurones with peripheral input from group II muscle afferents was analyzed after intracellular injection of horseradish peroxidase (HRP). The neurones were located in Rexed's laminae III-V overlying the pudendal (Onuf's) motor nucleus. The interneurones had medium sized elongated somata and dendrites projecting radially. All of the interneurones were funicular neurones and fell into two categories depending on whether their axons ran within the dorsal part of the lateral funiculus (DLF; n = 7) or within the ventral funiculus, or the ventral part of the lateral funiculus (VF or VLF; n = 4). The latter were located more rostrally. Within the DLF similar proportions of stem axons and secondary axonal branches descended and ascended. Within the VF and VLF all of the axons ascended. Collaterals of axons running in the DLF arborized primarily within the dorsal horn and the intermediate zone; none were found to approach the motor nuclei. In contrast, collaterals of axons running in the VF/VLF arborized in both the intermediate zone and the ventral horn and passed close to the motor nuclei. We conclude that sacral interneurones with group II input are morphologically nonhomogenous and that only those located most rostrally might have direct actions upon motoneurones. Both the axonal projections and the input (from group II but not from group I muscle afferents and from skin afferents) of sacral interneurones indicate that they are homologous to dorsal horn group II interneurones in the midlumbar segments. They appear, however, to form part of more local neuronal networks than their midlumbar counterparts.

Afferent Pathways↗
PubMed1994

Central processing of sex pheromone, host odour, and oviposition deterrent information by interneurons in the antennal lobe of female Spodoptera littoralis (Lepidoptera: Noctuidae).

Physiological and anatomical characteristics of antennal lobe interneurons in female Spodoptera littoralis (Boisd.) were investigated using intracellular recording and staining techniques. Responses of local interneurons and projection neurons to female sex pheromone components, host plant odours, and behaviourally active oviposition deterrents were recorded. We found local interneurons and projection neurons that responded specifically to only one or two of the tested odours, but we also found less specific cells, and neurons that responded to most of the tested odourants. These findings show that there are not only specific olfactory pathways in female moths up to the protocerebral level, but also that integration can begin in the antennal lobe. No correlation was found between the degree of specificity of either local interneurons or projection neurons and their respective morphological characteristics. Specialized and unspecialized local interneurons arborized throughout the antennal lobe. Specialized and unspecialized projection neurons had uniglomerular arborizations in the antennal lobe and sent their axons to the calyces of the mushroom body, and to the lateral horn of the protocerebrum. One specific projection neuron had multiglomerular arborizations and projected only to the lateral horn of the protocerebrum. Projection neurons arborizing in the glomeruli closest to the entrance of the antennal nerve always responded to pheromone components. No other correlations were found between the arborization pattern of projection neurons in the antennal lobe or in the protocerebrum and their response characteristics. The sensitivity of local interneurons and projection neurons was in the same range as that of receptor neurons in olfactory sensilla on the antennae, suggesting a much lower convergence in the central nervous system in females than in the pheromone-processing pathway in males.

Animals↗
PubMed1995

Crayfish brain interneurons that converge with serotonin giant cells in accessory lobe glomeruli.

Freshwater crayfish have well-developed olfactory systems with an array of receptors that project exclusively to areas in the brain that are functionally specialized for the processing of odors. The accessory lobes are large bilateral areas of neuropil that are anatomically associated with the olfactory lobes. The accessory lobes receive no primary afferents and do not contain the endings of motor efferents; thus, their role in olfaction is still obscure. Intracellular dye filling of interneurons in the deutocerebral commissure in the crayfish brain has shown that they end bilaterally in glomeruli in the accessory lobes, have cell somata in a dorsal cluster medial to the olfactory lobes, and have unilateral projections to the deutocerebral commissure neuropil. Each deutocerebral commissure interneuron has only 6 to 15 output glomeruli in each accessory lobe and does not share glomeruli with other deutocerebral commissure interneurons. The deutocerebral commissure interneurons converge with the dorsal giant serotonin neurons in the accessory lobe glomeruli. Deutocerebral commissure interneurons can be separated into classes according to their projections to the protocerebrum, central body, and deutocerebrum. Physiological responses of the deutocerebral commissure interneurons following photic stimulation of the eyes and electrical stimulation of the second antennae lead to the conclusion that the deutocerebral commissure represents an input to the accessory lobes from the protocerebral neuropils and that visual and tactile inputs are included in the processing performed in the accessory lobes.

Animals↗
PubMed1995

The development of interneurons in the chick embryo spinal cord following in vivo treatment with retinoic acid.

To investigate the role of retinoic acid (RA) in the development of interneurons in the spinal cord, we examined the expression of cellular retinoic acid binding protein type I (CRABP I). The earliest developing interneurons in the chick spinal cord can be divided into two major groups: circumferential (C) neurons and primitive longitudinal (PL) neurons. In brachial segments, both types of interneurons began to express CRABP I at stage (st.) 13+ of the V. Hamburger and H.L. Hamilton (1951, J. Morphol. 88:49-92) stage series, which is before the onset of axonogenesis. Subsequently, with the onset of axonal outgrowth, C neurons and PL neurons expressed CRABP I in their cell bodies, axons, and growth cones. The expression of CRABP I was developmentally regulated. CRABP I immunoreactivity gradually decreased after st. 36 (embryonic day [E] 10) such that no interneurons expressed this protein by E21. The transient expression of CRABP I during a period of intensive axonal growth suggested that RA may be involved in the development of interneurons. To test this idea, we implanted an all-trans RA-containing ion exchange bead into either rostral segments of the spinal cord at st. 12-13 or into caudal segments at st. 15-16, all stages that are well before the appearance of CRABP-I-positive neurons in these segments. In the RA-treated spinal cord, increased numbers of pyknotic cells were found predominantly in dorsal regions, presumably reflecting the death of neuroepithelial cells, C neurons and premigratory neural crest cells. Surviving C neurons in the RA-treated spinal cord extended their axons ventrally toward the floor plate as in control embryos. PL neurons also projected their axons rostrally or caudally in the RA-treated spinal cord, similarly to control embryos. However, the proportion of caudally projecting PL neurons was significantly increased in segments rostral to the RA-containing bead. These results suggest that RA may regulate the survival and axonal orientation (directionality) of subpopulations of spinal interneurons.

Animals↗