Morphological phenotyping of enteric neurons using neurofilament immunohistochemistry renders chemical phenotyping more precise in porcine ileum

The aim of the study was: (1) to test the suitability of neurofilament (NF) immunohistochemistry for representing the shapes of morphologically defined neuron types in the pig ileum myenteric plexus, (2) to estimate the proportions of these neuron types as related to the whole myenteric neuron population and (3) to demonstrate the usefulness of a refined morphological classification of enteric neurons on the paradigm of calcitonin gene-related peptide (CGRP)-immunoreactive neurons. So far, immunoreactivity for this peptide was supposed to be present in the pig enteric nervous system only in type II neurons. Ileal whole mounts of two pigs were stained with the cuprolinic blue (CB) method and, thereafter, incubated with an antibody pool against NF proteins (70, 160 and 210 kDa), visualised with a fluorochrome-tagged secondary antibody. The structural representation of morphologically defined myenteric neuron types typical for pig ileum (Stach I, II, IV, V and VI) was equivalent to their silver impregnated image, as demonstrated in previous studies. Counts of CB-stained neurons revealed between 2,526 and 2,662 neurons per square centimetre in one pig and between 2,027 and 2,763 in the other. As related to these total neuron numbers, the proportions of type I neurons were 1.7% and 1.5%, of type II neurons 7.2% and 7.9%, of type IV neurons 1.9% and 2.4%, of type V neurons 1.1% and 1.5%, and of type VI neurons 1.3% each. These values are generally comparable with those estimated earlier on silver impregnated material. Double labelling for NF and CGRP indicated that CGRP-immunoreactive smooth contoured neurons with long processes could be subdivided into two distinct morphological neuron types, i.e. type II and type V. We conclude that NF immunohistochemistry is an appropriate tool for representation of morphologically defined enteric neuron types in the pig. Combination of this technique with immunohistochemistry for neuroactive substances may be useful for making both morphological and chemical classification schemes mutually more precise.     

Calcitonin gene-related peptide in morphologically well-defined type II neurons of the enteric nervous system in the porcine small intestine

The morphological classification of the different neuronal cell types is generally accepted and expanded by us; nevertheless, immunohistochemically and electrophysiologically the existence of clear-cut categories of enteric neurons is frequently questioned. The immunohistochemical results demonstrated in this study, however, provide the first direct link between a morphological type of enteric neuron and an immunohistochemical staining for a distinct peptide. Evidence demonstrates that calcitonin gene-related peptide occurs in only one morphologically defined type of neuron, viz., in type II neurons, and can therefore be regarded as a 'marker peptide' for type II neurons. Hence, the present immunohistochemical findings illustrate the validity of the morphological classification of the enteric neurons.     

Ultrastructure of neurosecretory cells in the pars intercerebralis of Rhodnius prolixus (Hemiptera).

Six neuron types are distinguished in the pars intercerebralis of the starved fifth instar of Rhodnius prolixus. All neuron types contain electron dense secretory granules derived from Golgi complexes which are of characteristic size and morphology in each type. The neuron types are not thought to represent stages in a secretory cycle. The variety of neuron types described is related to that revealed by staining sections of the same cells with paraldehyde fuchsin. Active synthesis of neurosecretory granules continues throughout starvation and the lysosomal system appears to be involved in the continual degradation of secretory granules. Some of the variations in granule morphology observed may be a consequence of granule fusion and the importance of cytoplasmic events in the development of neurosecretory granules is discussed.     

An update to Hippocampome.org by integrating single-cell phenotypes with circuit function in vivo

Understanding brain operation demands linking basic behavioral traits to cell-type specific dynamics of different brain-wide subcircuits. This requires a system to classify the basic operational modes of neurons and circuits. Single-cell phenotyping of firing behavior during ongoing oscillations in vivo has provided a large body of evidence on entorhinal–hippocampal function, but data are dispersed and diverse. Here, we mined literature to search for information regarding the phase-timing dynamics of over 100 hippocampal/entorhinal neuron types defined in Hippocampome.org. We identified missing and unresolved pieces of knowledge (e.g., the preferred theta phase for a specific neuron type) and complemented the dataset with our own new data. By confronting the effect of brain state and recording methods, we highlight the equivalences and differences across conditions and offer a number of novel observations. We show how a heuristic approach based on oscillatory features of morphologically identified neurons can aid in classifying extracellular recordings of single cells and discuss future opportunities and challenges towards integrating single-cell phenotypes with circuit function.

By integrating single-cell firing behavior during in vivo oscillations with morphological and molecular neuron classification in vitro, this study standardizes a framework for interpreting the functional roles of distinct circuit modules and ascribing extracellular recordings to identified neuron types.     

Mapping study of Achatina giant neurons sensitive to molluscan peptides

1. Mapping studies of the Achatina identifiable neuron types sensitive to the following 6 molluscan peptides were examined under current-clamp.2. These were Ser-Mytilus inhibitory peptide (Ser-MIP), catch-relaxing peptide (CARP), oxytocin, small cardioactive peptide_b (SCP_b), α-bag cell peptide (α-BCP) and egg-laying hormone (ELH).3. These peptides at 10⁻³ M (3 × 10⁻⁴ M for ELH), with 0.5% Fast Green, were applied locally to the neuron to be tested by pneumatic pressure ejection (2 kg/cm² and 400 msec in duration).4. Ser-MIP showed the inhibitory (hyperpolarizing) effects on the majority of neuron types tested.5. CARP also produced inhibition of the 3 neuron types out of 16 types tested.6. Oxytocin had an excitatory effect on two neuron types.7. SCP_b showed excitatory effects on 4 neuron types: the membrane conductance of 1 neuron type, d-RPeAN, measured under voltage-clamp was reduced by the peptide.8. α-BCP showed no effect.9. ELH produced slight inhibition of the 2 neuron types.     

Non-traditional large neurons in the granular layer of the cerebellar cortex

The granular layer of the cerebellar cortex is composed of two groups of neurons, the granule neurons and the so-called large neurons. These latter include the neuron of Golgi and a number of other, lesser known neuron types, generically indicated as non-traditional large neurons. In the last few years, owing to the development of improved histological and histochemical techniques for studying morphological and chemical features of these neurons, some non-traditional large neurons have been morphologically well characterized, namely the neuron of Lugaro, the synarmotic neuron, the unipolar brush neuron, the candelabrum neuron and the perivascular neuron. Some types of non-traditional large neurons may be involved in the modulation of cortical intrinsic circuits, establishing connections among neurons distributed throughout the cortex, and acting as inhibitory interneurons (i.e., Lugaro and candelabrum neurons) or as excitatory ones (i.e., unipolar brush neuron). On the other hand, the synarmotic neuron could be involved in extrinsic circuits, projecting to deep cerebellar nuclei or to another cortex regions in the same or in a different folium. Finally, the perivascular neuron may intervene in the intrinsic regulation of the cortex microcirculation.     

Towards a supervised classification of neocortical interneuron morphologies

Background

The challenge of classifying cortical interneurons is yet to be solved. Data-driven classification into established morphological types may provide insight and practical value.

Results

We trained models using 217 high-quality morphologies of rat somatosensory neocortex interneurons reconstructed by a single laboratory and pre-classified into eight types. We quantified 103 axonal and dendritic morphometrics, including novel ones that capture features such as arbor orientation, extent in layer one, and dendritic polarity. We trained a one-versus-rest classifier for each type, combining well-known supervised classification algorithms with feature selection and over- and under-sampling. We accurately classified the nest basket, Martinotti, and basket cell types with the Martinotti model outperforming 39 out of 42 leading neuroscientists. We had moderate accuracy for the double bouquet, small and large basket types, and limited accuracy for the chandelier and bitufted types. We characterized the types with interpretable models or with up to ten morphometrics.

Conclusion

Except for large basket, 50 high-quality reconstructions sufficed to learn an accurate model of a type. Improving these models may require quantifying complex arborization patterns and finding correlates of bouton-related features. Our study brings attention to practical aspects important for neuron classification and is readily reproducible, with all code and data available online.

     

Clonal analysis of Drosophila antennal lobe neurons: diverse neuronal architectures in the lateral neuroblast lineage

The antennal lobe (AL) is the primary structure in the Drosophila brain that relays odor information from the antennae to higher brain centers. The characterization of uniglomerular projection neurons (PNs) and some local interneurons has facilitated our understanding of olfaction; however, many other AL neurons remain unidentified. Because neuron types are mostly specified by lineage and temporal origins, we use the MARCM techniques with a set of enhancer-trap GAL4 lines to perform systematical lineage analysis to characterize neuron morphologies, lineage origin and birth timing in the three AL neuron lineages that contain GAL4-GH146-positive PNs: anterodorsal, lateral and ventral lineages. The results show that the anterodorsal lineage is composed of pure uniglomerular PNs that project through the inner antennocerebral tract. The ventral lineage produces uniglomerular and multiglomerular PNs that project through the middle antennocerebral tract. The lateral lineage generates multiple types of neurons, including uniglomeurlar PNs, diverse atypical PNs, various types of AL local interneurons and the neurons that make no connection within the ALs. Specific neuron types in all three lineages are produced in specific time windows, although multiple neuron types in the lateral lineage are made simultaneously. These systematic cell lineage analyses have not only filled gaps in the olfactory map, but have also exemplified additional strategies used in the brain to increase neuronal diversity.     

An electric analogue of the neuron

An electric analogue model of the neuron is described. The model neuron is composed of the active units which simulate the electric behaviors of the active loci of the membrane of a neuron. The active units include the model axon and the model synapses of six different types (the ordinary, incremental and decremental onse each having the excitatory and the inhibitory types). The properties of the models are described.     

Gamma oscillations as a mechanism for selective information transmission

In the past decades, many studies have focussed on the relation between the input and output of neurons with the aim to understand information processing by neurons. A particular aspect of neuronal information, which has not received much attention so far, concerns the problem of information transfer when a neuron or a population of neurons receives input from two or more (populations of) neurons, in particular when these (populations of) neurons carry different types of information. The aim of the present study is to investigate the responses of neurons to multiple inputs modulated in the gamma frequency range. By a combination of theoretical approaches and computer simulations, we test the hypothesis that enhanced modulation of synchronized excitatory neuronal activity in the gamma frequency range provides an advantage over a less synchronized input for various types of neurons. The results of this study show that the spike output of various types of neurons [i.e. the leaky integrate and fire neuron, the quadratic integrate and fire neuron and the Hodgkin–Huxley (HH) neuron] and that of excitatory–inhibitory coupled pairs of neurons, like the Pyramidal Interneuronal Network Gamma (PING) model, is highly phase-locked to the larger of two gamma-modulated input signals. This implies that the neuron selectively responds to the input with the larger gamma modulation if the amplitude of the gamma modulation exceeds that of the other signals by a certain amount. In that case, the output of the neuron is entrained by one of multiple inputs and that other inputs are not represented in the output. This mechanism for selective information transmission is enhanced for short membrane time constants of the neuron.     

A stimulus-dependent connectivity analysis of neuronal networks

We present an analysis of interactions among neurons in stimulus-driven networks that is designed to control for effects from unmeasured neurons. This work builds on previous connectivity analyses that assumed connectivity strength to be constant with respect to the stimulus. Since unmeasured neuron activity can modulate with the stimulus, the effective strength of common input connections from such hidden neurons can also modulate with the stimulus. By explicitly accounting for the resulting stimulus-dependence of effective interactions among measured neurons, we are able to remove ambiguity in the classification of causal interactions that resulted from classification errors in the previous analyses. In this way, we can more reliably distinguish causal connections among measured neurons from common input connections that arise from hidden network nodes. The approach is derived in a general mathematical framework that can be applied to other types of networks. We illustrate the effects of stimulus-dependent connectivity estimates with simulations of neurons responding to a visual stimulus. This research was supported by the National Science Foundation grants DMS-0415409 and DMS-0748417.     

The state of gustatory neural coding

Debates on gustatory neural coding have been dominated by asmall number of fundamental issues since the inception of thefield in 1941. Three of these are discussed in this review:(1) are there basic tastes? (2) are there gustatory neuron types?(3) is the code for a taste read simultaneously across all participatingneurons (across-fiber patterning), or is it confined to a selectivechannel composed of cells of one type (labeled-line or channeling)?No conclusions are drawn regarding (1), primarily because auniversal definition of ‘basic tastes’ is lacking.It is concluded that gustatory neuron types are likely to existafter reviewing the issue from multiple perspectives and discoveringrecurring indications of neuron types from several. A firm conclusion,however, also awaits a widely accepted definition of what constitutesa neuron type. Issue (3) cannot yet be resolved for lack ofdefinitive data, specifically whether the discharges of inhibited,unresponsive, or weakly responsive cells add to (signal) ordetract from (noise) the neural code for a tastant.     

Role of type-specific neuron properties in a spinal cord motor network

Recent recordings from spinal neurons in hatchling frog tadpoles allow their type-specific properties to be defined. Seven main types of neuron involved in the control of swimming have been characterized. To investigate the significance of type-specific properties, we build models of each neuron type and assemble them into a network using known connectivity between: sensory neurons, sensory pathway interneurons, central pattern generator (CPG) interneurons and motoneurons. A single stimulus to a sensory neuron initiates swimming where modelled neuronal and network activity parallels physiological activity. Substitution of firing properties between neuron types shows that those of excitatory CPG interneurons are critical for stable swimming. We suggest that type-specific neuronal properties can reflect the requirements for involvement in one particular network response (like swimming), but may also reflect the need to participate in more than one response (like swimming and slower struggling). Action Editor: Eberhard E. Fetz     

Molecular biology of amyotrophic lateral sclerosis: insights from genetics

Amyotrophic lateral sclerosis (ALS) is a paralytic disorder caused by motor neuron degeneration. Mutations in more than 50 human genes cause diverse types of motor neuron pathology. Moreover, defects in five Mendelian genes lead to motor neuron disease, with two mutations reproducing the ALS phenotype. Analyses of these genetic effects have generated new insights into the diverse molecular pathways involved in ALS pathogenesis. Here, we present an overview of the mechanisms for motor neuron death and of the role of non-neuronal cells in ALS.     

Communicating synapses: types and functional interpretation. Exceptions to Cajal's neuron theory.

The neurons of the dorsal periaqueductal nucleus of the mesencephalon and their synaptic contacts were observed under a transmission electron microscope. We found various types of synapses which constituted an exception to Cajal's neuron theory (law of neuron independence). Some of these synapses had an open communicating or continuity 'passage' between the presynaptic bouton of a neuron (first neuron) and the postsynaptic portion of another neuron (second neuron). The 'communicating' passage (located in the synaptosome) is formed by the continuity of the presynaptic and postsynaptic membrane, and its limits or rims are the reflexion points of the membranes. When only two neurons intervene they could be termed 'simple communicating synapses'. We found three types: I = communicating axosomatic synapses; II = communicating axodendritic synapses, and III = communicating axoaxonic synapses'. When three neurons intervene in the synaptic contact, they could be termed 'complex communicating synapses'. In these, the first and second neurons form a normal synapse, but the lateral portion of the presynaptic bouton of the first neuron also enters into contact with a third neuron, with which it establishes an open communicating or continuity passage. The points of these passages are collateral to the synapse, and may be in the presynaptic or pre-postsynaptic portions simultaneously, communicating collaterally with the third neuron. We found a further three types: IV = complex communicating axosomatic and dendritic synapses; V = complex communicating axoaxonic and somatic synapses, and VI = complex communicating axodendritic and double-somatic synapses. It is suggested that communicating synapses may constitute an exception to Cajal's neuron theory, representing functional states for the acceleration, retardation or modulation of the synaptic function. The neurotransmitters would pass en masse through the communicating passage and the depolarization wave would pass through the rims without being retarded. In the simple communicating synapses, their action would be intensifying. In the complex communicating synapses, their action would be modulating or retarding, since the collateral communicating passage would function as an 'escape valve' through which part of the impulse reaching the presynaptic bouton would escape.     

Identification of thermosensory and olfactory neuron-specific genes via expression profiling of single neuron types

Most C. elegans sensory neuron types consist of a single bilateral pair of neurons, and respond to a unique set of sensory stimuli. Although genes required for the development and function of individual sensory neuron types have been identified in forward genetic screens, these approaches are unlikely to identify genes that when mutated result in subtle or pleiotropic phenotypes. Here, we describe a complementary approach to identify sensory neuron type-specific genes via microarray analysis using RNA from sorted AWB olfactory and AFD thermosensory neurons. The expression patterns of subsets of these genes were further verified in vivo. Genes identified by this analysis encode 7-transmembrane receptors, kinases, and nuclear factors including dac-1, which encodes a homolog of the highly conserved Dachshund protein. dac-1 is expressed in a subset of sensory neurons including the AFD neurons and is regulated by the TTX-1 OTX homeodomain protein. On thermal gradients, dac-1 mutants fail to suppress a cryophilic drive but continue to track isotherms at the cultivation temperature, representing the first genetic separation of these AFD-mediated behaviors. Expression profiling of single neuron types provides a rapid, powerful, and unbiased method for identifying neuron-specific genes whose functions can then be investigated in vivo.     

A Complete Developmental Sequence of a Drosophila Neuronal Lineage as Revealed by Twin-Spot MARCM

Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain.