Morphology and physiological properties of interneurons in the olfactory midbrain of the crayfish

1. Intracellular recording and staining was used to characterize neurons in the crayfish (Procambarus clarkii) brain that respond to chemical stimuli applied to the major olfactory organs, the antennules. 2. Two distinct morphological types of neurons that have major projections in the olfactory lobes (OLs) of the brain were characterized anatomically (Figs. 1, 2, 3; Table 2) and physiologically (Figs. 4, 5, 6; Table 3). 3. Different individual neurons of one type, with similar 'tree-like' projections in the OLs, have somata distributed in at least 5 different cell body clusters of the brain (Fig. 3) and link different subsets of neuropilar lobes through their distributed arbors (Fig. 1, Table 2). 4. Excitatory, inhibitory and mixed responses were recorded in different neurons when odorant mixtures or individual components of these mixtures were applied to the antennules. Response spectra to individual components were broad and overlapping, but not identical in the neurons tested (Fig. 4; Table 3). Mixture interactions appear to be additive in most of the neurons that we tested, but evidence was obtained for mixture suppression in several cases (Fig. 6). 5. Most of the neurons recorded in this study responded only to stimulation of the ipsilateral antennule (Fig. 5), although subthreshold activity to stimuli applied contralaterally was recorded in several neurons that were strongly excited by ipsilateral stimuli. 6. Chemoresponsive neurons without projections in OL's that have all of their branches confined to the brain, or that project an axon in the circumesophageal connective, are described (Fig. 7).     

Immunolocalisation of crustacean-SIFamide in the median brain and eyestalk neuropils of the marbled crayfish

Crustacean-SIFamide (GYRKPPFNGSIFamide) is a novel neuropeptide that was recently isolated from crayfish nervous tissue. We mapped the localisation of this peptide in the median brain and eyestalk neuropils of the marbled crayfish (Marmorkrebs), a parthenogenetic crustacean. Our experiments showed that crustacean-SIFamide is strongly expressed in all major compartments of the crayfish brain, including all three optic neuropils, the lateral protocerebrum with the hemiellipsoid body, and the medial protocerebrum with the central complex. These findings imply a role of this peptide in visual processing already at the level of the lamina but also at the level of the deeper relay stations. Immunolabelling is particularly strong in the accessory lobes and the deutocerebral olfactory lobes that receive a chemosensory input from the first antennae. Most cells of the olfactory globular tract, a projection neuron pathway that links deuto- and protocerebrum, are labelled. This pathway plays a central role in conveying tactile and olfactory stimuli to the lateral protocerebrum, where this input converges with optic information. Weak labelling is also present in the tritocerebrum that is associated with the mechanosensory second antennae. Taken together, we suggest an important role of crustacean-SIFamidergic neurons in processing high-order, multimodal input in the crayfish brain.     

Physiological changes of premotor nonspiking interneurons in the central compensation of eyestalk posture following unilateral sensory ablation in crayfish

We investigated how the physiological characteristics and synaptic activities of nonspiking giant interneurons (NGIs), which integrate sensory inputs in the brain and send synaptic outputs to oculomotor neurons innervating eyestalk muscles, changed after unilateral ablation of the statocyst in order to clarify neuronal mechanisms underlying the central compensation process in crayfish. The input resistance and membrane time constant in recovered animals that restored the original symmetrical eyestalk posture 2 weeks after operation were significantly greater than those immediately after operation on the operated side whereas in non-recovered animals only the membrane time constant showed a significant increase. On the intact side, both recovered and non-recovered animals showed no difference. The frequency of synaptic activity showed a complex pattern of change on both sides depending on the polarity of the synaptic potential. The synaptic activity returned to the bilaterally symmetrical level in recovered animals while bilateral asymmetry remained in non-recovered ones. These results suggest that the central compensation of eyestalk posture following unilateral impairment of the statocyst is subserved by not only changes in the physiological characteristics of the NGI membrane but also the activity of neuronal circuits presynaptic to NGIs.     

Structural re-evaluation of the neurosecretory system in the crayfish eyestalk

Summary The topography of the neurosecretory system in the decapod eyestalk has not been precisely delineated with light microscopy. Cobalt iontophoresis and electron microscopy have proved useful in clarifying the microstructure of this system. The sinus gland (sg) of the crayfish eyestalk consists of aggregated axon terminals which end at or near the blood space, lontophoresing cobalt back through the cut base of the sinus glands reveals proximal cell bodies in the eyestalk only in the X organ (Xo) region. Electron microscopy demonstrates that axons from about 115 neurosecretory cell bodies in the Xo form the Xo-sg tract. Intermingled with these Xo somata are smaller non-neurosecretory cell bodies which do not send axons into the sinus gland. One of these exhibits catecholamine fluorescence. Backfilling also reveals a second group of fibres which run from the brain along the optic tract and into the sinus gland. These brain-sg fibres are smaller in diameter than Xo-sg axons and lack neurosecretory vesicles. From these fibres collaterals extend into the eyestalk neuropil, especially in the proximity of the visual elements. The possible function of these non-neurosecretory processes within the sinus gland is discussed.This work was supported by a National Research Council of Canada grant     

Habituation of mechanoreceptive interneurons in the crayfish

The effect of repetition of sensory stimuli was studied on the responses of mechanoreceptive interneurons in the optic tract of the crayfish (Procambarus bouvieri (Ortmann)). The number of spikes recorded from a given unit gradually decreased during a train of stimuli. The decrease showed a negative exponential time course, with a curvature dependent upon the frequency of stimulation, the intensity of stimuli, and the hour of the day. Habituation is selective for the intensity and rate of stimulation, and the particular spot of receptive field stimulated. Locomotor excitation results in a dimminution of the rate of decay. The effect of a single train of stimuli when leading to pronounced habituation may persist for periods longer than 24 hr.     

Multimodal responses of the nonspiking giant interneurons in the brain of the crayfish Procambarus clarkii

Three pairs of nonspiking giant interneurons (NGIs; G1, G2, and G3) of the crayfish brain responded with depolarizing and hyperpolarizing graded potentials to body tilt in roll to the ipsi- and contralateral sides in the dark. The higher and the larger the angle of body tilt, the larger was the amplitude of the geotactic responses. In ipsilaterally statocystectomized animals, all the NGIs responded with hyperpolarizing potentials only to the contralateral side-down tilt, whereas in contralaterally statocystectomized animals, they responded with depolarizing potentials only to the ipsilateral side-down tilt. In bilaterally statocystectomized animals, none of the NGIs responded to body tilt in the dark, but in the presence of an overhead light, they exhibited depolarizing and hyperpolarizing potentials in response to body tilt to the ipsi-and contralateral sides, respectively. All the NGIs responded with depolarizing and hyperpolarizing graded potentials to illumination of the contra- and ipsilateral eyes, respectively. The amplitude of these visual responses, however, varied in association with the amplitude of the geotactic response produced by body tilt. These results indicate that the NGIs integrate the sensory inputs from eyes and statocysts and that the interaction between sensory inputs from the left and right sensory organs with either the same modality or with different modalities enhance the directional sensitivity of NGIs as premotoneurons in the compensatory oculomotor system.     

Modification of statocyst input to local interneurons by behavioral condition in the crayfish brain

Posture control by statocysts is affected by leg condition in decapod crustaceans. We investigated how, in the crayfish brain, the synaptic response of local interneurons to statocyst stimulation was affected by leg movements on and off a substratum. The magnetic field stimulation method permitted sustained stimulation of statocyst receptors by mimicking body rolling. The statocyst-driven local interneurons were classified into four morphological groups (Type-I–IV). All interneurons except Type-IV projected their dendritic branches to the parolfactory lobe of the deutocerebrum where statocyst afferents project directly. Type-I interneurons having somata in the ventral-paired lateral cluster responded invariably to statocyst stimulation regardless of the leg condition, whereas others having somata in the ventral-unpaired posterior cluster showed response enhancement or suppression, depending on the cell, during leg movements on a substratum, but no response change during free leg movements off the substratum. The synaptic responses of Type-II and IV interneurons were also affected differently by leg movements depending on the substratum condition, whereas those of Type-III remained unaffected. These findings suggest that the statocyst pathway in the crayfish brain is organized in parallel with local circuits that are affected by leg condition and those not affected.     

Secretory stages of individual CHH-producing cells in the eyestalk of the crayfish Astacus leptodactylus,determined by means of immunocytochemistry

Summary Immunocytochemical staining demonstrates striking differences in staining intensity among individual crustacean hyperglycemic hormone (CHH)-producing cells in the eyestalk of the crayfish Astacus leptodactylus. Based on these differences we arbitrarily subdivided the CHH-cells into three categories representing increasing immunoreactivity respectively: + cells, + + cells, and + + + cells. Electron microscopic investigations reveal that these differences in immunostaining are correlated with differences in the numerical density of the neurosecretory granules in the cytoplasm and that these may reflect differences in activity among the CHH-cells. Morphometric analyses at the light- and electron-microscopic levels indicate that the three distinguished categories of immunopositive cells represent different stages in the CHH-synthesizing process of the cells. The results of the present study demonstrate the application of the PAP-technique at the light-microscopic level as a method to obtain information pertaining to the dynamics of secretory activity of the CHH-cells.     

Morphological and physiological identification of medulla interneurons in the visual system of the tiger beetle larva

The morphology of visual interneurons in the tiger beetle larva was identified after recording their responses. Stained neurons were designated as either medulla or protocerebral neurons according to the location of their cell bodies. Medulla neurons were further subdivided into three groups. Afferent medulla neurons extended processes distally in the medulla neuropil and a single axon to the brain through the optic nerve. They received their main input from stemmata on the ipsilateral side. Two distance-sensitive neurons, near-by sensitive and far-sensitive neurons, were also identified. Atypical medulla neurons extended their neurites distally in the medulla and proximally to the brain, as afferent medulla neurons, but their input patterns and the shapes of their spikes differed from afferent neurons. Protocerebral neurons sent a single axon to the medulla neuropil. They spread collateral branches in the posterior region of the protocerebrum on its way to the medulla neuropil. They received main input from stemmata on the contralateral side. Medulla intrinsic neurons did not extend an axon to the brain, and received either bilateral or contralateral stemmata input only. The input patterns and discharge patterns of medulla neurons are discussed with reference to their morphology.     

Responses of non-spiking giant interneurons to substrate tilt in the crayfish, with special reference to multisensory control in the compensatory eyestalk movement system

In the brain of the intact crayfish, three pairs of non-spiking giant interneurons (G1, G2, G3; NGIs) scarcely responded to substrate tilt about the longitudinal axis of the body either in the dark or in the presence of an overhead light. However, when the statolith was removed, these NGIs responded with depolarizing and hyperpolarizing potentials respectively to upward movements of the ipsilateral legs (2nd–5th pereiopods) and upward movements of the contralateral legs produced by substrate tilt. The relationships between the polarity of the potential and the direction of movement in the contralateral legs were opposite to those in the ipsilateral legs. The amplitude of the responses was proportional to the frequency (0.5-0.05 Hz) and amplitude of tilting. When the legs were moved unilaterally, the NGIs responded with depolarizing and hyperpolarizing potentials to upward movements of the ipsilateral legs and to upward movements of the contralateral legs, respectively. When the legs were moved bilaterally in the same direction by upward or downward movement of the substrate, the NGIs scarcely responded to the leg movements. A hypothetical model is presented to account for the pathways of sensory inputs to the NGIs and the role of NGIs in compensatory oculomotor system.     

Habituation of mechanoreceptive interneurons in the crayfish.

The effect of repetition of sensory stimuli was studied on the responses of mechanoreceptive interneurons in the optic tract of the crayfish (Procambarus bouvieri (Ortmann)). The number of spikes recorded from a given unit gradually decreased during a train of stimuli. The decrease showed a negative exponential time course, with a curvature dependent upon the frequency of stimulation, the intensity of stimuli, and the hour of the day. Habituation is selective for the intensity and rate of stimulation, and the particular spot of receptive field stimulated. Locomotor excitation results in a dimminution of the rate of decay. The effect of a single train of stimuli when leading to pronounced habituation may persist for periods longer than 24 hr.     

Local interneurons in the swimmeret system of the crayfish

Summary We describe the structures and physiological properties of thirteen kinds of local interneurons in the swimmeret system of the crayfish,Pacifastacus leniusculus. Eight are unilateral, with processes confined to one side of the midline (Figs. 1, 2); five are bilateral, with processes on both sides of the ganglion (Fig. 6). All have most of their branches in the lateral neuropils. All of the unilateral local interneurons were nonspiking; two of the bilateral interneurons generate action potentials. Three kinds of unilateral interneurons could reset the bursting rhythm or could initiate bursting in quiescent nerve cords. Four others drove tonic firing of motor neurons. Four kinds of bilateral interneurons were premotor, and could affect the period and phase of both pattern generators in their ganglion. One unilateral and one bilateral interneuron were sensory interneurons. At least one bilateral interneuron received input from both pattern generators.Different premotor local interneurons function either in pattern generation, or in hemisegmental coordination of groups of motor neurons, or in bilateral synchronization of the ganglionic pairs of local pattern-generators for the swimmerets.Abbreviations G1. ganglion 1. - LN lateral neuropil - MT miniscule tract     

Physiological and morphological characterization of olfactory descending interneurons of the male silkworm moth, Bombyx mori

In order to understand the neural mechanisms of pheromone-oriented walking in male silkworm moths, Bombyxmori, we have characterized olfactory responses and three-dimensional structure of two clusters (Group-I, Group-II) of descending interneurons in the brain by intracellular recording and staining with lucifer yellow. Neurons were imaged with laser-scanning confocal microscopy. Group-I and Group-II descending interneurons were classified into three morphological types, respectively. In response to the sex pheromone, bombykol, Type-A Group-I descending interneurons showed characteristic flipflopping activity. The Group-I descending interneurons had dendritic arborizations in the lateral accessory lobe and varicose profiles in the posterior-lateral part of the suboesophageal ganglion where the dendritic arborizations of a neck motor neuron (i.e., cv1 NMN) reside. Other types of Group-I descending interneurons exhibited long-lasting suppression of firing. The pheromonal responses of Group-II descending interneurons fell into two classes: brief excitation and brief inhibition. Type-A Group-II descending interneurons showing brief excitation had blebby processes in the posterior-lateral part of the suboesophageal ganglion. Type-B and Type-C Group-II descending interneurons did not have varicose profiles there. Therefore, the neck motor neuron regulating head turning, which accompanies the pheromone-oriented walking, may be controlled by these two types, flipflop and phasic excitation, of descending activity patterns. Accepted: 2 November 1998     

Morphological and physiological properties of pheromone-triggered flipflopping descending interneurons of the male silkworm moth,Bombyx mori

1. The morphology of descending interneurons (DNs) which have arborizations in the lateral accessory lobe (LAL) of the protocerebrum, the higher order olfactory center, and have an axon in the ventral nerve cord (VNC), were characterized in the male silkworm moth, Bombyx mori.
2. Two clusters (group I, group II) of DNs which have arborizations mainly in the LALs were morphologically characterized. The axons of these DNs are restricted to the dorsal part of the each connective (Figs. 1–5).
3. Pheromonal responses of the group I and group II DNs were characterized. Flipflopping activity patterns, which have two distinct firing frequencies (high and low) in response to sequential pheromonal stimulation, were usually recorded (Figs.6–10).
4. Two types of flipflopping activity patterns were classified into those that had an antiphasic relationship (called the ‘FF’ type) between the left and right connectives and those with a synchronized relationship (‘ff’ type) (Figs. 8–12). We propose that some group II DNs show ‘FF’ flipflopping activity patterns (Fig. 10).
5. A state transition was usually elicited by less than 10 ng bombykol, the principal pheromone component. Extra impulses were elicited during constant light stimulation (Fig. 9).
6. Our results suggest that the LAL olfactory pathways might be important for producing flipflopping activity patterns (Fig. 11).

     

Sensory interneurons: some observations concerning the physiology and related structural significance of two cells in the crayfish brain

Two large interneurons in the crayfish brain which are sensitive to vibrational stimuli were injected with the fluorescent dye Procion Yellow. The dendritic branching profiles reflect the directional sensitivity of their respective mechanoreceptive fields on the cephalic appendages and integument. One interneuron branches exclusively on the contralateral side of the brain and receives monosynaptic input from the contralateral antenna; the second interneuron branches primarily on the ipsilateral side and is more sensitive to input from ipsilateral receptors although its receptive field is bilateral. The data suggest that these cells are primary and secondary sensory interneurons, respectively.     

Circadian oscillations of RPCH gene expression in the eyestalk of the crayfish Cherax quadricarinatus

The RPCH and β-actin cDNAs from the crayfish Cherax quadricarinatus were amplified, cloned and sequenced. The primary structure sequences of these cDNAs were compared to other members of the AKH/RPCH family. Fluctuations in the amount of the C. quadricarinatus RPCH and β-actin mRNAs, as cDNAs, were quantified every 3 h by RT-PCR. Single cosinor analysis supports the notion of β-actin and RPCH mRNA circadian behavior in animals subjected to 12 h:12 h light/dark regimes. In constant darkness RPCH mRNA concentration changes to ultradian cycles.     

Insulin receptors in the brain: Structural and physiological characterization

The present study was conducted to characterize insulin receptors and to determine the effects of insulin in synaptosomes prepared from adult rat brains. Binding of¹²⁵I-insulin to synaptosome insulin receptors was highly specific and time dependent: equilibrium binding was obtained within 60 minutes, and a t_1/2 of dissociation of 26 minutes. Cross-linking of¹²⁵I-insulin to its receptor followed by SDS-PAGE demonstrated that the apparent molecular weight of the alpha subunit of the receptor was 122,000 compared with 134,000 for the liver insulin receptor. In addition, insulin stimulated the dose-dependent phosphorylation of exogenous tyrosine containing substrate and a 95,000 MW plasma membrane associated protein, in a lectin-purified insulin receptor preparation. The membrane associated protein was determined to be the subunit of the insulin receptor. Incubation of synaptosomes with insulin caused a dose-dependent inhibition of specific sodium-sensitive [³H]norepinephrine uptake. Insulin inhibition of [³H]norepinephrine uptake was mediated by a decrease in active uptake sites without any effects in theK _m, and was specific for insulin since related and unrelated peptides influenced the uptake in proportion to their structural similarity with insulin. These observations indicate that synaptosomes prepared from the adult rat brain possess specific insulin receptors and insulin has inhibitory effects on norepinephrine uptake in the preparation.