Edward R. Perl

Correlations between neuronal morphology and electrophysiological features in the rodent superficial dorsal horn.

Relationships between the morphology of individual neurones of the spinal superficial dorsal horn (SDH), laminae I and II, and their electrophysiological properties were studied in spinal cord slices prepared from anaesthetized, free‐ranging hamsters. Tight‐seal, whole‐cell recordings were made with pipette microelectrodes filled with biocytin to establish electrophysiological characteristics and to label the studied neurones. Neurones were categorized according to location and size of the somata, the dendritic and axonal pattern of arborization, spontaneous synaptic potentials, evoked postsynaptic currents, pattern of discharge to depolarizing pulses and current‐voltage relationships. Data were obtained for 170 neurones; 13 of these had somata in lamina I and 157 in lamina II. Stimulation of the segmental dorsal root evoked a prompt excitatory response in almost every neurone sampled (161/166) with nearly 3/4 displaying putative monosynaptic EPSCs. The majority of neurones (133/170) fitted one of several distinctive morphological categories. To a considerable extent, neurones with a common morphological configuration and neurite disposition shared electrophysiological characteristics. Five of the 13 lamina I neurones were relatively large with extensive dendritic arborization in the horizontal dimension and a prominent axon directed ventrally and contralaterally. These presumptive ventrolateral projection neurones differed structurally and electrophysiologically from the other lamina I neurones, which had ipsilateral, locally arborizing axons and/or branches entering the dorsal lateral funiculus. One hundred and twenty lamina II neurones fitted one of five morphological categories: islet, central, medial‐lateral, radial or vertical. Central cells were further divided into three groups on functional features. We conclude that the spinal SDH comprises many types of neurones whose morphological characteristics are associated with specific functional features implying diversity in functional organization of the SDH and in its role as a major synaptic termination for thin primary afferent fibres.

Relationship of substance P to afferent characteristics of dorsal root ganglion neurones in guinea‐pig

1 The relationship between the afferent properties and substance P‐like immunoreactivity (SP‐LI) of L6 and SI dorsal root ganglion (DRG) neuronal somata was examined in anaesthetized guinea‐pigs. Glass pipette microelectrodes filled with fluorescent dyes were used to make intracellular recordings and to label DRG somata. The dorsal root conduction velocity (CV) and the afferent receptive properties of each unit were categorized according to criteria established in other species. Categories included a variety of low threshold mechanoreceptive classes, innocuous thermoreceptive and several nociceptive classes. Nociceptive units were further subdivided on the basis of CV and the locus of the receptive field (superficial cutaneous, deep cutaneous or subcutaneous). 2 SP‐LI was determined using the avidin–biotin complex method and the relative staining intensity determined by image analysis. The possible significance of labelling intensity is discussed. Clear SP‐LI appeared in twenty‐nine of 117 dye‐labelled neurones. All SP‐LI positive units with identified receptive properties were nociceptive but not all categories of nociceptors were positive. The intensity of SP‐LI labelling varied, often systematically, in relation to afferent properties. There was a tendency for nociceptive neurones with slower CVs and/or smaller cell bodies to show SP‐LI. 3 Nineteen of fifty‐one C fibre neurones showed SP‐LI. Fewer than half the C polymodal nociceptors (CPMs) were positive. The most intensely labelled units were the deep cutaneous nociceptors and some of the CPMs in glabrous skin. C low threshold mechanoreceptors and cooling‐sensitive units did not show SP‐LI. 4 Ten of sixty‐six A fibre neurones exhibited SP‐LI, including eight of sixteen Aδ nociceptors and two of fifteen Aα/β nociceptors. A fibre neurones exhibiting SP‐LI included seven of eight deep cutaneous mechanical nociceptors and some superficial cutaneous mechano‐heat nociceptors of hairy skin. In contrast, none of twenty superficial cutaneous A high threshold mechanoreceptor units or the thirty‐five A fibre low threshold units (D‐hair and other units) showed detectable SP‐LI. 5 We conclude that SP‐LI labelling in guinea‐pig DRG neurones is related to (a) afferent receptive properties, (b) the tissue in which the peripheral receptive terminals are located, (c) the CV and (d) the soma size.

A Specific Inhibitory Pathway between Substantia Gelatinosa Neurons Receiving Direct C-Fiber Input

The spinal substantia gelatinosa (SG) is a major termination region for unmyelinated (C) primary afferent fibers; however, how the input it receives from these sensory fibers is processed by SG neurons remains primarily a matter of conjecture. To gain insight on connections and functional interactions between intrinsic SG neurons, simultaneous tight-seal, whole-cell recordings were made from pairs of neurons in rat spinal cord slices to examine whether impulses in one cell generated synaptic activity in the other. Most SG neuron pairs sampled lacked synaptic interaction. Those showing a linkage included a recurring pattern consisting of a monosynaptic, bicuculline-sensitive inhibitory connection from an islet cell to a transient central neuron, each of which received direct excitatory input from different afferent C-fibers. This newly defined inhibitory circuit is postulated to represent a SG neural module by which a nociceptive C-fiber input to transient central cells is modified by other C-fiber messages.

Modular Organization of Excitatory Circuits between Neurons of the Spinal Superficial Dorsal Horn (Laminae I and II)

Neural circuitry of the spinal superficial dorsal horn (SDH) (laminae I and II) and its relationship to pain and other somatosensory phenomena remain poorly understood. To gain information on this issue, synaptic connections between identified SDH neurons were studied in rat spinal cord slices by simultaneous whole-cell recordings from pairs of cells. Both excitatory and inhibitory connections were noted. This report focuses on the observed excitatory linkages. Synaptic excitatory connections between SDH neurons proved highly selective and consistently were unidirectional. Two patterns repeatedly appeared (for neuron classification, see Materials and Methods) (Grudt and Perl, 2002). Lamina II central neurons, with dorsal root (DR) C-fiber input, monosynaptically excited lamina II vertical neurons with DR Aδ input. Lamina II outer vertical neurons with DR Aδ input monosynaptically excited lamina I neurons. Some of the postsynaptic lamina I cells were shown to project rostrally. In contrast to the usual case for connected neurons, in unconnected pairs, primary afferent input to the same type of neuron proved closely similar. Together, these observations indicate that the neural circuitry in the SDH, including its substantia gelatinosa (lamina II), has an explicit organization in which particular combinations of neurons comprise modules arranged to modify and transmit sensory information arriving from Aδ and C primary afferent fibers.

Expression of α2-adrenergic receptors in rat primary afferent neurones after peripheral nerve injury or inflammation

1 Immunocytochemistry with polyclonal antibodies directed against specific fragments of intracellular loops of α2A‐ and α2C‐adrenergic receptors (α2A‐AR, α2C‐AR) was used to explore the possibility that expression of these receptors in dorsal root ganglion (DRG) neurones of rat alters as a result of peripheral nerve injury or localized inflammation. 2 Small numbers of neurones with positive α2A‐AR immunoreactivity (α2A‐AR‐IR) were detected in DRG from normal animals or contralateral to nerve lesions. In contrast, after complete or partial sciatic nerve transection the numbers of ipsilateral L4 and L5 DRG somata expressing α2A‐AR‐IR sharply increased (>5‐fold). There was no discernible change in the number of DRG neurones exhibiting α2A‐AR‐IR innervating a region in association with localized chemically induced inflammation. 3 After nerve injury, double labelling with Fluoro‐Gold, a marker of retrograde transport from transected fibres, or by immunoreactivity for c‐jun protein, an indicator of injury and regeneration, suggested that many of the neurones expressing α2A‐AR‐IR were uninjured by the sciatic lesions. 4 In general the largest proportionate increase in numbers of neurones labelled by α2A‐AR‐IR after nerve lesions appeared in the medium‐large diameter range (31–40 μm), a group principally composed of cell bodies of low threshold mechanoreceptors. The number of small diameter DRG neurones labelled by α2A‐AR‐IR, a category likely to include somata of nociceptors, also increased but proportionately less. 5 Relatively few DRG neurones exhibited α2C‐AR‐IR; this population did not appear to change after either nerve lesions or inflammation. 6 These observations are considered in relation to effects of nerve injury on excitation of primary afferent neurones by sympathetic activity or adrenergic agents, sympathetically related neuropathy and reports of sprouting of sympathetic fibres in DRG.

Differential termination of large-diameter and small-diameter primary afferent fibers in the spinal dorsal gray matter as indicated by labeling with horseradish peroxidase

Abstract Primary afferent fibers, collaterals, and terminations in the spinal dorsal horn were labeled by the orthograde transport of horseradish peroxidase. Lesions of fine afferent fibers comprising the lateral division of dorsal rootlets resulted in the staining of almost no boutons in the marginal zone and substantia gelatinosa but of many boutons in the nucleus proprius. Lesions of large-diameter fibers comprising the medial division restricted staining to a very few boutons in the nucleus proprius while many were stained in the marginal zone and the substantia gelatinosa.

Selective excitation of neurons in the mammalian spinal dorsal horn by aspartate and glutamate in vitro: correlation with location and excitatory input

The electrical activity of mammalian dorsal horn neurons was recorded with pipette microelectrodes in an in vitro spinal cord slice preparation with dorsal roots intact. Addition of relatively low concentrations of aspartate or glutamate to the superfusion solution or through the recording pipette with small iontophoretic currents excited only a subset of neurons. The majority of these excited neurons were located in the superficial dorsal horn (Rexeds laminae I and II) and a preponderance were excited by the C-fiber components of dorsal root volleys. These findings are consistent with the idea that aspartate or glutamate may function as a synaptic mediator for some neurons terminating in the superficial dorsal horn.

Sensitization of high threshold receptors with unmyelinated (C) afferent fibers.

Publisher Summary Polymodal nociceptors are a dominant cutaneous C-fiber sense organ of mammals. They are characterized by relatively high thresholds for mechanical and thermal stimuli and respond vigorously only to noxious levels of heat, mechanical, and chemical stimuli. Noxious stimulation of the receptive field of polymodal nociceptors, particularly by heat, results in an enhanced responsiveness, lowered threshold and the development of background activity; these changes have been labeled sensitization. Sensitization develops in less than one minute and can last for hours. The process of sensitization, while exhibiting localization is capable of spreading. Demonstrable evidence of spread of sensitization appears related to the degree of skin damage. Sensitization develops in skin preparations perfused with artificial solutions not containing proteins or protein fragments. Serotonin, antihistaminic, and antiprostaglandin agents do not cause major modifications in the development of sensitization.

Morphological and Physiological Features of a Set of Spinal Substantia Gelatinosa Neurons Defined by Green Fluorescent Protein Expression

The spinal substantia gelatinosa (SG) is known to be involved in the manipulation of nociceptive and thermal primary afferent input; however, the interrelationships of its neuronal components are poorly understood. As a step toward expanding understanding, we took a relatively unique approach by concentrating on a set of SG neurons selectively labeled by green fluorescent protein (GFP) in a transgenic mouse. These GFP-expressing SG neurons prove to have homogenous morphological and electrophysiological properties, are systematically spaced in the SG, contain GABA, receive C-fiber primary afferent input, and upregulate c-Fos protein in response to noxious stimuli. Together, the properties established for these GFP-labeled neurons are consistent with a modular SG organization in which afferent activity related to nociception or other C-fiber signaling are subject to integration/modulation by repeating, similar circuits of neurons.

Calcitonin gene‐related peptide immunoreactivity and afferent receptive properties of dorsal root ganglion neurones in guinea‐pigs

To establish the afferent receptive properties of lumbosacral dorsal root ganglion (DRG) neurones that express calcitonin gene‐related peptide (CGRP), intracellular recordings were made with fluorescent dye‐filled electrodes in deeply anaesthetised young guinea‐pigs. After determination of neuronal functional properties, dye was injected into the soma. CGRP‐like immunoreactivity (CGRP‐LI) was examined on histological sections of dye‐marked neurones. Fourteen of 34 C‐fibre neurones showed CGRP‐LI. These included 10/21 C‐fibre nociceptive neurones. All C‐polymodal nociceptors in glabrous (n= 4) but none in hairy skin (n= 4) were positive. Positive C‐fibre high threshold mechanoreceptive (HTM) units had receptive fields in dermal or deeper tissue. Four (n= 6) unresponsive or unidentified C‐fibre units were positive. Neither C‐fibre cooling sensitive (n= 4) nor C‐fibre low threshold mechanoreceptive (LTM) units (n= 3) had CGRP‐LI. Six of 23 A‐fibre nociceptive cells were positive including one Aα/β unit. Three of these positive cells had epidermal and three had dermal/deep receptive fields. Three of 36 A‐fibre LTM units exhibited CGRP‐LI; all were Aα/β‐fibre G hair units. All glabrous skin and muscle spindle units and in hairy skin slowly adapting and field units, and some G‐hair units lacked CGRP‐LI. CGRP‐LI stained fibres were found in tissues containing receptive fields of positive DRG neurones: glabrous skin, near hair follicles and in skeletal muscle. A few substance P‐labelled neurones did not exhibit CGRP‐LI and vice versa. Thus CGRP expression was detected in under half the nociceptive neurones, was not limited to nociceptive neurones and apart from receptive properties was also related to location/depth in the tissues of a DRG neurones peripheral terminals.