Modelling nonlinear integration of synaptic signals by neurones

Neurones with active conductance on dendrites integrate synaptic signals and modulate generation of axon spikes in a nonlinear way. Owing to experimental difficulties, modelling provides invaluable insight for the comprehension of neurone behaviour particularly when dendrites are excitable. We used experimental data obtained for the Anterior Gastric Receptor neurone (AGR neurone), which controls the lobster gastric mill activity, to derive a set of partial differential equations for the membrane voltage. Simulation showed that upon varying the intensity of stimulation on the dendrite, the response pattern between dendrites and axon activity continuously changes. In addition, when only half of the dendritic tree is active, axon firing exhibits regular oscillations and bursting activity. We discuss these results in relation with the experimental work done on the AGR neurone.     

Localization of the enhanced input to cockroach giant interneurons after partial deafferentation

The ventral giant interneurons (GIs) in the cockroach have two distinct dendritic fields: a small one ipsilateral to the soma, and a larger, contralateral field from which the axon arises. The major input to these GIs is from the cercus on the axon side; when this cercus is ablated in the last instar before the adult stage, input from the other cercus becomes more effective within 30 days (Vardi and Camhi, 1982b). I wished to determine if the input from the intact, soma-ipsilateral cercus contacted the GIs purely ipsilaterally and if EPSPs at this site were larger in deafferented animals. Consistent with earlier anatomical findings, intracellular recordings from the GI somata showed that the majority of cercal inputs synapse on their own side of the ganglion in normal animals. This was evidenced by differences in the size and shape of the synaptic potentials evoked from the two cerci and by the presence of large EPSPs after a ganglion had been split along the midline. Unitary EPSPs produced by stimulation of single, identified cercal afferents, ipsilateral to the soma, were compared between normal and deafferented animals. Column "h" afferents were chosen because they make a large contribution to the receptive fields of GIs 1 and 2 after ablation of the contralateral cercus. In addition, the arbors of these afferents, when stained with cobalt, did not cross the ganglionic midline in normal animals. Unitary EPSPs recorded in GI 2 were significantly larger in the deafferented animals. There was, however, no significant change in the size of EPSPs in GI 1. Nevertheless, the results from GI 2 suggest that partial deafferentation in the central nervous system can increase the efficacy of synapses distant from the locus of denervation.     

Control of prothoracic gland activity in larvae of Galleria mellonella

Prothoracic glands of last instar wax moth larvae maintain spontaneous secretory activity both in decapitated larvae and in isolated abdomens into which they have been transplanted, as judged by their ability to induce secretion of a new cuticle. Their activity is hormonally stimulated by the brain and inhibited by the prothoracic and mesothoracic ganglia. The subesophageal ganglion seems to suppress the inhibitory influence of the thoracic ganglia. The prothoracic glands of larvae decapitated at different times during the last instar all respond to brain implantation, and this response does not change when brains are implanted at increasing intervals after decapitation. The prothoracotropic activity of the isolated brain is highest in brains of pupae and adults but is relatively and consistently low in brains of last instar larvae. The results demonstrate that the control of prothoracic glands is a complex process governed by the nervous integration of various stimuli.     

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS^1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries³. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved⁴. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth^5-7.Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection^6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons⁹.     

Neuronal mechanisms of learning in an in vitro Aplysia preparation: sites other than the sensory-motor neuron synapse are involved

Classical conditioning of the gill withdrawal reflex can be demonstrated in two different in vitro Aplysia preparations. The data obtained show that as conditioning of the gill withdrawal reflex proceeds there are changes in synaptic efficacy at the central sensory-motor neurone synapse. These changes in synaptic efficacy, however, are not necessary nor are they sufficient for the observed changes in gill reflex behaviour. Changes must be occurring at other loci within the nervous system to mediate the associative learning. We hypothesized, based on data obtained from one type of in vitro preparation, that changes occur in the ability of the motor neurone to elicit a gill withdrawal response as a result of classical conditioning training. In order to test this hypothesis we depolarized an identified gill motor neurone before and after classical conditioning and found that the motor neurone's ability to elicit a gill movement was facilitated following classical conditioning training. In control preparations that received an explicitly unpaired stimulus paradigm (which does not lead to classical conditioning of the reflex) there was a decrease in the efficacy of a gill motor neurone to elicit a gill withdrawal response. There are a number of possible sites within the integrated central (CNS) and peripheral (PNS) nervous systems where changes could occur to bring about the alterations in motor neurone efficacy. Our results suggest that changes in neuronal activity which underlie learning occur at multiple sites within the nervous system and that a complete understanding of the mechanisms of associative learning can only be obtained when all of these sites are taken into account.     

Reliable, responsive pacemaking and pattern generation with minimal cell numbers: the crustacean cardiac ganglion

Investigations of the electrophysiology of crustacean cardiac ganglia over the last half-century are reviewed for their contributions to elucidating the cellular mechanisms and interactions by which a small (as few as nine cells) neuronal network accomplishes extremely reliable, rhythmical, patterned activation of muscular activity-in this case, beating of the neurogenic heart. This ganglion is thus a model for pacemaking and central pattern generation. Favorable anatomy has permitted voltage- and space-clamp analyses of voltage-dependent ionic currents that endow each neuron with the intrinsic ability to respond with rhythmical, patterned impulse activity to nonpatterned stimulation. The crustacean soma and initial axon segment do not support impulse generation but integrate input from stretch-sensitive dendrites and electrotonic and chemically mediated synapses on axonal processes in neuropils. The soma and initial axon produce a depolarization-activated, calcium-mediated, sustained potential, the "driver potential," so-called because it drives a train of impulses at the "trigger zone" of the axon. Extreme reliability results from redundancy and the electrotonic coupling and synaptic interaction among all the neurons. Complex modulation by central nervous system inputs and by neurohormones to adjust heart pumping to physiological demands has long been demonstrated, but much remains to be learned about the cellular and molecular mechanisms of action. The continuing relevance of the crustacean cardiac ganglion as a relatively simple model for pacemaking and central pattern generation is confirmed by the rapidly widening documentation of intrinsic potentials such as plateau potentials in neurons of all major animal groups. The suite of ionic currents (a slowly inactivating calcium current and various potassium currents, with variations) observed for the crustacean cardiac ganglion have been implicated in or proven to underlie a majority of the intrinsic potentials of neurons involved in pattern generation.     

Interactions between p-tyramine, m-tyramine, or beta-phenylethylamine and dopamine on single neurones in the cortex and caudate nucleus of the rat

p-Tyramine, applied to cortical and caudate neurones with weak iontophoretic currents (0-10 nA), did not usually cause any alteration of base-line firing rate. However, neuronal responses to dopamine (DA) during such weak applications of p-tyramine were greatly enhanced. Cortical neurone responses to noradrenaline (NA) were similarly potentiated, but both cortical and caudate neurone responses to alpha-aminobutyric acid were unaffected by p-tyramine. In addition, weak background applications of DA which did not affect cell firing rate were also without effect on the neuronal responses to the standard application of DA. The responses of cortical neurones to DA were also potentiated by m-tyramine and beta-phenylethylamine applied with weak cationic currents. The results may suggest that trace amines can enhance NA and DA transmission in the central nervous system.     

L��essor des sciences du neurone au xx e si��cle

In the late 19th century, the introduction of the neurone concept led to vivid oppositions in many fields of enquiry, especially in the physiology of the nervous system. In Great Britain, novel research programs (Sherrington and Adrian) supplanted the general common hostility to conceive of the neurone as a general and fundamental physiological element. These new paths of research led to a unique neuronal physiology awarded the Nobel Prize for physiology or medicine in 1932. This first form of neuro-physiology spread abroad and came under the attack of American physiologists concerning the functional role of the neurone soma vs the more fundamental and hypothetical function of the axon. During the 1930s and the 1940s, a series of polemics progressively died out with the establishment of the fundamental bases of a new and international neuronal physiology, which led to the rise of neuroscience after the Second World War.     

Extracellular matrix and perineuronal nets in CNS repair

A perineuronal net (PNN) is a layer of lattice-like matrix which enwraps the surface of the soma and dendrites, and in some cases the axon initial segments, in sub-populations of neurons in the central nervous system (CNS). First reported by Camillo Golgi more than a century ago, the molecular structure and the potential role of this matrix have only been unraveled in the last few decades. PNNs are mainly composed of hyaluronan, chondroitin sulfate proteoglycans, link proteins, and tenascin R. The interactions between these molecules allow the formation of a stable pericellular complex surrounding synapses on the neuronal surface. PNNs appear late in development co-incident with the closure of critical periods for plasticity. They play a direct role in the control of CNS plasticity, and their removal is one way in which plasticity can be re-activated in the adult CNS. In this review, we examine the molecular components and formation of PNNs, their role in maturation and synaptic plasticity after CNS injury, and the possible mechanisms of PNN action.     

Tarsal chemoreception in the polyphagous grasshopper Schistocerca americana: behavioural assays, sensilla distributions and electrophysiology

ABSTRACT Behavioural and electrophysiological responses of Schistocerca americana (Drury) (Orthoptera: Acrididae) to chemical stimulation of the tarsi were investigated. Using restrained insects, differences in leg-waving behaviour were observed following stimulation by sucrose and nicotine hydrogen tartrate (NHT), compared to control stimulations by water. Furthermore, free-walking insects were able to detect NHT on leaf surfaces, resulting in leg-raising to avoid tarsal contact.
SEM studies showed the presence of numerous peg chemoreceptor sensilla on the ventral surface of the tarsus. Tip recordings from such pegs showed activity from up to three chemosensitive neurones, plus a mechanoreceptor neurone. Stimulation by NaCl and KC1 elicited similar responses from two or three neurones in all sensilla tested, with increased firing rates at higher concentrations. Sucrose caused an increase in firing rate in few sensilla. In such cases several neurones were stimulated, and there was no evidence of a specific neurone sensitive to sucrose. In contrast, NHT elicited rapid firing in a single neurone, which was not sensitive to NaCl. Stimulation by NHT also inhibited the activity of the NaCl-sensitive neurones.
Possible mechanisms for chemical discrimination in S. americana tarsi are compared with those previously proposed for grasshopper mouthpart sensilla, and the significance of a NHT-sensitive neurone in tarsal sensilla is discussed.     

Correlations between juvenile hormone esterase activity,ecdysone titre and cellular reprogramming in Galleria mellonella

Juvenile hormone esterase (JHE) activity, ecdysone titre, and developmental competence of the epidermis were determined in last instar larvae and pupae of Galleria mellonella. Haemolymph JHE activity reaches a peak before increases are observed in ecdysone titre both during larval-pupal and pupal-adult metamorphosis. JHE activity is low during the penultimate larval instar although general esterase activity is relatively high. In last instar larvae two ecdysone peaks are noted after the increase in JHE activity. Furthermore, epidermal cell reprogramming occurs just after the increase in haemolymph JHE activity and possibly before the first increase in ecdysone titre. This was tested by injection of high doses of β-ecdysone into last instar larvae of different ages resulting in rapid cuticle deposition. Reprogramming occurred if the resulting cuticle was of the pupal type. These correlative observations may increase our understanding of the relative importance of an ecdysone surge in the absence of JH in reprogramming of the insect epidermis.     

Locust solid cuticle—A time sequence of mechanical properties

Studies on locust femur solid cuticle indicate that there is a continuum of biomechanically important properties from one instar to the next, and that there is a constant ratio of stiffness to mass which is only interrupted at ecdysis. The biochemical nature of both larval and adult solid cuticle is interpreted in terms of both the tangent modulus and the breaking strength of the femur cuticle. The mechanical behaviour of the different layers in the cuticle is discussed.     

Modulation of motoneuronal firing behavior after spinal cord injury using intraspinal microstimulation current pulses: a modeling study.

We simulated the effects of delivering focal electrical stimuli to the central nervous system to modulate the firing rate of neurons and alleviate motor disorders. Application of these stimuli to the spinal cord to reduce the increased excitability of motoneurons and resulting spasticity after spinal cord injury (SCI) was examined by means of a morphologically detailed computer model of a spinal motoneuron. High-frequency sinusoidal and rectangular pulses as well as biphasic charge-balanced and charge-imbalanced pulses were examined. Our results suggest that suprathreshold high-frequency sinusoidal or rectangular current pulses could inactivate the Na+ channels in the soma and initial segment, and block action potentials from propagating through the axon. Subthreshold biphasic charge-imbalanced pulses reduced the motoneuronal firing rate significantly (up to approximately 25% reduction). The reduction in firing rate was achieved through stimulation-induced hyperpolarization generated in the first node of Ranvier. Because of their low net DC current, these pulses could be tolerated safely by the tissue. To deliver charge-imbalanced pulses with the lowest net DC current and induce the largest reduction in motoneuronal firing rate, we studied the effect of various charge-imbalanced pulse parameters. Short pulse durations were found to induce the largest reduction in firing rate for the same net DC level. Subthreshold high-frequency sinusoidal and rectangular current pulses and low-frequency biphasic charge-balanced pulses, on the other hand, were ineffective in reducing the motoneuronal firing rate. In conclusion, the proposed electrical stimulation paradigms could provide potential rehabilitation interventions for suppressing the excitability of neurons to reduce the severity of motor disorders after injury to the central nervous system.     

Zipper encodes a putative integral membrane protein required for normal axon patterning during Drosophila neurogenesis.

During the development of the central nervous system, Drosophila embryo axons become organized in a stereo-typed fasciculation pattern. We have found that the zipper (zip) gene, initially identified on the basis of a defective larval cuticle in zip mutant embryos, is possibly involved in the establishment or maintenance of the axon pattern during the late stages of neurogenesis. The zip wild-type gene is expressed in the developing nervous system. It codes for a putative integral membrane protein. Both the molecular features of zipper and its biological effect in the nervous system of mutants suggest that zipper is an essential component for cell surface interactions involved in axon patterning, and that the cuticle phenotype of zip mutants is dependent on the primary defects observed in the nervous system.     

Neuropilin signalling in vessels,neurons and tumours

The neuropilins NRP1 and NRP2 are transmembrane proteins that regulate many different aspects of vascular and neural development. Even though they were originally identified as adhesion molecules, they are most commonly studied as co-receptors for secreted signalling molecules of the class 3 semaphorin (SEMA) and vascular endothelial growth factor (VEGF) families. During nervous system development, both classes of ligands control soma migration, axon patterning and synaptogenesis in the central nervous system, and they additionally help to guide the neural crest cell precursors of neurons and glia in the peripheral nervous system. Both classes of neuropilin ligands also control endothelial cell behaviour, with NRP1 acting as a VEGF-A isoform receptor in blood vascular endothelium and as a semaphorin receptor in lymphatic valve endothelium, and NRP2 promoting lymphatic vessel growth induced by VEGF-C. Here we provide an overview of neuropilin function in neurons and neural crest cells, discuss current knowledge of neuropilin signalling in the vasculature and conclude with a summary of neuropilin roles in cancer.     

The growth of axons in three-dimensional astrocyte cultures

The environment of the adult central nervous system (CNS) does not support axon regeneration. We have been unable to replicate this behaviour using monolayer cultures of glia, so we have developed a technique for three dimensional culture of glial cells. We have examined the growth of axons from embryonic and postnatal retina and dorsal root ganglia (DRG's) through purified three-dimensional astrocyte cultures. Neither postnatal DRG's nor adult retina were able to grow axons through astrocytes from cultures 3 weeks or more old, although some DRG axons grew in astrocyte cultures which were 10 days or less old. However axons from embryonic DRG's and retina grew axons profusely into even elderly astrocyte cultures. All the tissues grew axons into three-dimensional Schwann cell cultures. The behaviour of axons in three-dimensional glial cultures therefore reproduces the behaviour of axons in vivo.     

The cuticle proteins of Drosophila melanogaster: stage specificity

Five stage-specific cuticles are produced during the development of Drosophila. Urea-soluble proteins were extracted from each developmental stage and compared by gel electrophoresis. Proteins from first and second instar cuticle are identical except for minor differences in two proteins. Each subsequent stage, third instar, pupa, and adult, has a unique set of cuticle proteins. Qualitative changes within stages are seen in proteins from third instar and adult cuticle. Third instar cuticle proteins can be divided into “early” [proteins 2a, 3, 4, 5, 7, and 8] and “late” [proteins 2 and 1] groups. Adult cuticle proteins change in relative amounts during pharate adult development and change mobility at eclosion. The lower abdominal pupal cuticle lacks a protein found in the pupal cuticle covering the head and thorax. Cuticle proteins from each stage are immunologically related. Nonetheless, electrophoretic variants of three larval proteins do not affect any major changes in the electrophoretic mobility of proteins from other stages. We propose that each stage (except first and second instar) has proteins encoded by discrete genes.     

Response properties of the prothoracic AN2 auditory interneurone to model calling songs in the cricket Gryllus bimaculatus

Sound processing properties for calling song (CS) models, as described for the prothoracic L3 auditory neurone in Acheta domesticus, are investigated for the homologous auditory neurone 2 (AN2) in female Gryllus bimaculatus De Geer. AN2 of G. bimaculatus responds selectively to the syllable period (SP) of models of a male CS. The selectiveness of this response parallels the selectivity of phonotaxis females perform in response to the same SPs. Both, the responses of AN2 and female behaviour show clear interindividual variability. The SP‐selective responses of AN2 result from an SP‐dependent reduction in the spiking to subsequent syllables of the model CSs, measured as the percentage decrement. This SP‐dependent response does not primarily result from inbuilt properties of the AN2 membrane. Rather, it is dependent on inhibitory input to the AN2. However, clear inhibitory postsynaptic potentials in dendritic recordings of the AN2 are not encountered. This immediate response of AN2 to CSs is followed by an increased rate of tonic firing between stimulus CSs, which is termed the prolonged response, and is dependent on the carrier frequencies that make up the male CSs. With stimulation on the contralateral side of the soma of AN2s, more than 50% of AN2s exhibit a prolonged response. However, with stimulation from the ipsilateral side of the soma, most AN2s exhibit a prolonged response. The prolonged response of AN2 at 5 kHz may be even more sensitive than the immediate response. Thus, the AN2 neurone could provide a basis for phonotaxis that is selective for both the SPs and the carrier frequencies of potentially attractive calling songs.     

Organization of motoneurones in the prothoracic ganglion of the cockroach Periplaneta americana (L.).

The location within the prothoracic ganglion of neurone somata with axons in identified peripheral nerves is examined by the cobalt iontophoresis technique. Axons are filled with cobalt by diffusion through their cut ends and the cobalt is then precipitated as the black sulphide inside the neurone. It is assumed that neurones with axons in peripheral nerves and somata in central ganglia are either motor or neuro-secretory. Fifteen nerves are examined and maps of the location of somata with axons in each nerve are presented. The axon distribution in peripheral nerves of three common inhibitory neurones is described. Dendritic morphology of one common inhibitory neurone and two coxal depressor motoneurones is illustrated. It is proposed that some individual neurones can be reliably identified from their soma dimensions and location within the ganglion. The number of motoneurones with somata in the prothoracic ganglion and their homology with cells in the other thoracic ganglia are discussed.     

Ion Channel Density Regulates Switches between Regular and Fast Spiking in Soma but Not in Axons

The threshold firing frequency of a neuron is a characterizing feature of its dynamical behaviour, in turn determining its role in the oscillatory activity of the brain. Two main types of dynamics have been identified in brain neurons. Type 1 dynamics (regular spiking) shows a continuous relationship between frequency and stimulation current (f-I_stim) and, thus, an arbitrarily low frequency at threshold current; Type 2 (fast spiking) shows a discontinuous f-I_stim relationship and a minimum threshold frequency. In a previous study of a hippocampal neuron model, we demonstrated that its dynamics could be of both Type 1 and Type 2, depending on ion channel density. In the present study we analyse the effect of varying channel density on threshold firing frequency on two well-studied axon membranes, namely the frog myelinated axon and the squid giant axon. Moreover, we analyse the hippocampal neuron model in more detail. The models are all based on voltage-clamp studies, thus comprising experimentally measurable parameters. The choice of analysing effects of channel density modifications is due to their physiological and pharmacological relevance. We show, using bifurcation analysis, that both axon models display exclusively Type 2 dynamics, independently of ion channel density. Nevertheless, both models have a region in the channel-density plane characterized by an N-shaped steady-state current-voltage relationship (a prerequisite for Type 1 dynamics and associated with this type of dynamics in the hippocampal model). In summary, our results suggest that the hippocampal soma and the two axon membranes represent two distinct kinds of membranes; membranes with a channel-density dependent switching between Type 1 and 2 dynamics, and membranes with a channel-density independent dynamics. The difference between the two membrane types suggests functional differences, compatible with a more flexible role of the soma membrane than that of the axon membrane.