Visualization of spinal afferent innervation in the mouse colon by AAV8-mediated GFP expression.

BACKGROUND: Primary afferent neurons whose cell bodies reside in thoracolumbar and lumbosacral dorsal root ganglia (DRG) innervate colon and transmit sensory signals from colon to spinal cord under normal conditions and conditions of visceral hypersensitivity. Histologically, these extrinsic afferents cannot be differentiated from intrinsic fibers of enteric neurons because all known markers label neurons of both populations. Adeno-associated virus (AAV) vectors are capable of transducing DRG neurons after intrathecal administration. We hypothesized that AAV-driven overexpression of green fluorescent protein (GFP) in DRG would enable visualization of extrinsic spinal afferents in colon separately from enteric neurons. METHODS: Recombinant AAV serotype 8 (rAAV8) vector carrying the GFP gene was delivered via direct lumbar puncture. Green fluorescent protein labeling in DRG and colon was examined using immunohistochemistry. KEY RESULTS: Analysis of colon from rAAV8-GFP-treated mice demonstrated GFP-immunoreactivity (GFP-ir) within mesenteric nerves, smooth muscle layers, myenteric plexus, submucosa, and mucosa, but not in cell bodies of enteric neurons. Notably, GFP-ir colocalized with CGRP and TRPV1 in mucosa, myenteric plexus, and globular-like clusters surrounding nuclei within myenteric ganglia. In addition, GFP-positive fibers were observed in close association with blood vessels of mucosa and submucosa. Analysis of GFP-ir in thoracolumbar and lumbosacral DRG revealed that levels of expression in colon and L6 DRG appeared to be related. CONCLUSIONS & INFERENCES: These results demonstrate the feasibility of gene transfer to mouse colonic spinal sensory neurons using intrathecal delivery of AAV vectors and the utility of this approach for histological analysis of spinal afferent nerve fibers within colon.

Identification of dorsal root ganglion neurons that innervate the common bile duct of rats.

Pain originating in the bile duct is common and many patients who have suffered from it report that it is one of the most intense forms of pain that they have experienced. Many uncertainties remain about the mechanisms underlying pain originating in the bile duct. For example, the dorsal root ganglion (DRG) neurons that give rise to the sensory innervation of the common bile duct (CBD) have not been identified and examined in any species. The goal of the present study was to determine the number, distribution, and size of DRG neurons that innervate the CBD in rats. Injections of WGA-HRP or CTB-HRP were restricted to the lumen of the bile duct. Injections of WGA-HRP labeled a mean number of about 500 DRG neurons bilaterally throughout all thoracic and upper lumbar levels. Injections of CTB-HRP labeled smaller numbers of DRG neurons. Application of colchicine onto the surface of the CBD reduced the number of cells labeled following injections of WGA-HRP into the lumen of the CBD by roughly 86%, suggesting that tracer had not spread in large amounts out of the CBD and labeled afferent fibers in other tissues. Approximately 85% of the neurons labeled with WGA-HRP had cell bodies that were classified as small; the remainder were medium in size. Injections of CTB-HRP labeled cell bodies of varying sizes, including a few large diameter cell bodies. These results indicate that a large number of primarily small DRG cells, located bilaterally at many segmental levels, provide a rich innervation of the common bile duct.

Cytotoxic targeting of isolectin IB4-binding sensory neurons.

The isolectin I-B4 (IB4) binds specifically to a subset of small sensory neurons. We used a conjugate of IB4 and the toxin saporin to examine in vivo the contribution of IB4-binding sensory neurons to nociception. A single dose of the conjugate was injected unilaterally into the sciatic nerve of rats. The treatment resulted in a permanent selective loss of IB4-binding neurons as indicated by histological analysis of dorsal root ganglia, spinal cord, and skin from treated animals. Behavioral measurements showed that 7-10 days after the injection, conjugate-treated rats had elevated thermal and mechanical nociceptive thresholds. However, 21 days post-treatment the nociceptive thresholds returned to baseline levels. These results demonstrate the utility of the IB4-saporin conjugate as a tool for selective cytotoxic targeting and provide behavioral evidence for the role of IB4-binding neurons in nociception. The decreased sensitivity to noxious stimuli associated with the loss of IB4-binding neurons indicates that these sensory neurons are essential for the signaling of acute pain. Furthermore, the unexpected recovery of nociceptive thresholds suggests that the loss of IB4-binding neurons triggers changes in the processing of nociceptive information, which may represent a compensatory mechanism for the decreased sensitivity to acute pain.

Projections from the marginal zone and deep dorsal horn to the ventrobasal nuclei of the primate thalamus.

It has been concluded recently that if a projection from the marginal zone to the ventral posterior lateral (VPL) nucleus exists, it is sparse. Given the importance of the marginal zone in nociception, this conclusion has raised doubts about the significance of the role of the ventrobasal complex in nociception. We have reexamined this projection using injections of the retrograde tracer, cholera toxin subunit B, into one side of the lateral thalamus in macaque monkeys. The injections were confined to the ventrobasal complex (with minimal spread to adjacent nuclei that do not receive spinal projections) in two animals. Many retrogradely labeled neurons were found in lamina I (as well as in lamina V) of the contralateral spinal and medullary dorsal horn. The results are consistent with the view that neurons in the marginal zone contribute prominently to the spinothalamic and trigeminothalamic projections to the VPL and ventral posterior medial (VPM) nuclei. This pathway is likely to be important for the sensory-discriminative processing of nociceptive information with respect to the location and intensity of painful stimuli.

Position of spinothalamic tract axons in upper cervical spinal cord of monkeys.

Percutaneous upper cervical cordotomy continues to be performed on patients suffering from several types of severe chronic pain. It is believed that the operation is effective because it cuts the spinothalamic tract (STT), a primary pathway carrying nociceptive information from the spinal cord to the brain in humans. In recent years, there has been controversy regarding the location of STT axons within the spinal cord. The aim of this study was to determine the locations of STT axons within the spinal cord white matter of C2 segment in monkeys using methods of antidromic activation. Twenty lumbar STT cells were isolated. Eleven were classified as wide dynamic range neurons, six as high-threshold cells, and three as low-threshold cells. Eleven STT neurons were recorded in the deep dorsal horn and nine in superficial dorsal horn. The axons of the examined neurons were located at antidromic low-threshold points (<30 microA) within the contralateral lateral funiculus of C2. All low-threshold points were located ventral to the denticulate ligament, within the lateral half of the ventral lateral funiculus (VLF). None were found in the dorsal half of the lateral funiculus. The present findings support our previous suggestion that STT axons migrate ventrally as they ascend the length of the spinal cord. Also, the present findings indicate that surgical cordotomies that interrupt the VLF in C2 likely disrupt the entire lumbar STT.

Locations of spinothalamic tract axons in cervical and thoracic spinal cord white matter in monkeys.

The spinothalamic tract (STT) is the primary pathway carrying nociceptive information from the spinal cord to the brain in humans. The aim of this study was to understand better the organization of STT axons within the spinal cord white matter of monkeys. The location of STT axons was determined using method of antidromic activation. Twenty-six lumbar STT cells were isolated. Nineteen were classified as wide dynamic range neurons and seven as high-threshold cells. Fifteen STT neurons were recorded in the deep dorsal horn (DDH) and 11 in superficial dorsal horn (SDH). The axons of 26 STT neurons were located at 73 low-threshold points (<30 microA) within the lateral funiculus from T(9) to C(6). STT neurons in the SDH were activated from 33 low-threshold points, neurons in the DDH from 40 low-threshold points. In lower thoracic segments, SDH neurons were antidromically activated from low-threshold points at the dorsal-ventral level of the denticulate ligament. Neurons in the DDH were activated from points located slightly ventral, within the ventral lateral funiculus. At higher segmental levels, axons from SDH neurons continued in a position dorsal to those of neurons in the DDH. However, axons from neurons in both areas of the gray matter were activated from points located in more ventral positions within the lateral funiculus. Unlike the suggestions in several previous reports, the present findings indicate that STT axons originating in the lumbar cord shift into increasingly ventral positions as they ascend the length of the spinal cord.

Physiological studies of spinohypothalamic tract neurons in the lumbar enlargement of monkeys.

Recent anatomic results indicate that a large direct projection from the spinal cord to the hypothalamus exists in monkeys. The aim of this study was to determine whether the existence of this projection could be confirmed unambiguously using electrophysiological methods and, if so, to determine the response characteristics of primate spinohypothalamic tract (SHT) neurons. Fifteen neurons in the lumbar enlargement of macaque monkeys were antidromically activated using low-amplitude current pulses in the contralateral hypothalamus. The points at which antidromic activation thresholds were lowest were found in the supraoptic decussation (n = 13) or in the medial hypothalamus (n = 2). Recording points were located in the superficial dorsal horn (n = 1), deep dorsal horn (n = 10), and intermediate zone (n = 4). Each of the 12 examined neurons had cutaneous receptive fields on the ipsilateral hindlimb. All neurons responded exclusively or preferentially to noxious stimuli, suggesting that the transmission of nociceptive information is an important role of primate SHT axons. Twelve SHT neurons were also antidromically activated from the thalamus. In all cases, the antidromic latency from the thalamus was shorter than that from the hypothalamus, suggesting that the axons pass through the thalamus then enter the hypothalamus. These results confirm the existence of a SHT in primates and suggest that this projection may contribute to the production of autonomic, neuroendocrine, and emotional responses to noxious stimuli in primates, possibly including humans.

Immunohistochemical localization of delta opioid receptors in peripheral tissues.

The distribution of delta opioid receptor (DOR) immunoreactivity (ir) was examined in various peripheral tissues of Sprague-Dawley rats and macaque monkeys, including glabrous and hairy skin, corneas, eyelids, and the lip. DOR-ir was observed in all tissues examined. In addition to the presence of DOR-immunoreactive fibers in subcutaneous nerve bundles and the papillary dermis, we report the existence of positively labeled fibers and terminals in close association with peripheral structures not traditionally assigned a primarily nociceptive function, such as hair follicles, glandular apparatus, and blood vessels. In every case, staining was restricted to small-diameter axons that appeared to terminate as free nerve endings. To further classify DOR-immunoreactive fibers, we examined the extent of colocalization between DOR and three commonly used neuronal subtype markers; tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), and RT-97, a monoclonal antibody which preferentially labels neurons with myelinated axons. Additional double-labeling experiments using the nonspecific neuronal marker Protein Gene Product 9.5 were performed in glabrous skin to determine the percentage of total fiber count that displayed DOR-ir. No colocalization was observed between DOR and RT-97, indicating that DOR-ir is localized to unmyelinated axons. In addition, DOR colocalized with CGRP, but did not colocalize with TH. Taken together, these data support the hypothesis that delta opioid receptors in peripheral tissues are associated with sensory fibers, but not with the terminals of postganglionic sympathetic neurons.

A method for improving the accuracy of stereotaxic procedures in monkeys using implanted fiducial markers in CT scans that also serve as anchor points in a stereotaxic frame.

We developed a relatively inexpensive method for stereotaxic placement of electrodes or needles in the brains of monkeys. Steel balls were affixed to the skulls of monkeys. These balls served as fiducial markers and were also used as points at which the monkey's skull was held in a modified stereotaxic apparatus. Computed tomography (CT) was used to establish the location of an injection target with respect to the fiducial markers. A computer program related the CT coordinates to stereotaxic coordinates. These were used to direct an electrode marker toward a target in the hypothalamus. With the marker left in place, the monkey was removed from the stereotaxic frame and a second CT scan was performed. Corrections for errors in marker placement were made and retrograde tracers were injected. This procedure was found to be more accurate and reliable than conventional stereotaxic procedures. The accuracy and repeatability of the technique were also established using a phantom model of a monkey's skull. Two important advantages of this method are that animals can be repeatedly placed into the stereotaxic frame in precisely the same position and that there are many opportunities during the procedure to check for and correct errors.

Spinal NK1 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation.

Hyperalgesia is a characteristic of inflammation and is mediated, in part, by an increase in the excitability of spinal neurons. Although substance P does not appear to mediate fast synaptic events that underlie nociception in the spinal cord, it may contribute to the hyperalgesia and increased excitability of spinal neurons during inflammation induced by complete Freund's adjuvant. We examined the role of endogenous substance P in changes in the excitability of spinal neurons during adjuvant-induced, peripheral inflammation by determining the effect of a selective NK1 receptor antagonist (RP67580) on the nociceptive flexor reflex in adult rats. Experiments were conducted 2 or 3 days after injection of adjuvant. Animals exhibited moderate thermal hyperalgesia at this time. The flexor reflex was evoked by electrical stimulation of the sural nerve and was recorded in the ipsilateral hamstring muscles. The flexor reflex ipsilateral to the inflamed hindpaw was enhanced approximately two-fold compared to the flexor reflex evoked in untreated animals as determined by the number of potentials and the duration of the reflex. The enhanced reflex in adjuvant-treated animals was most likely due to an increase in the excitability of spinal interneurons because short-latency activity in the hamstring muscles did not differ between untreated animals and adjuvant-treated animals following electrical stimulation of the L5 dorsal root or the nerve innervating the muscle with a stimulus that was 1.3-1.5 times the threshold for excitation of A-fibers. Intrathecal administration of RP67580 (2.3 and 6.8 nmol) attenuated the flexor reflex evoked in adjuvant-treated animals, but had no effect in untreated animals. Intravenous or intraplantar injection of RP67580 (6.8 nmol) did not affect the flexor reflex in adjuvant-treated animals indicating a spinal action of the drug following intrathecal administration. RP68651, the enantiomer of RP67580, was without effect at doses up to 6.8 nmol, indicating that the effects of comparable doses of RP67580 were due to an action of the drug at NK1 receptors. However, intrathecal administration of 23 nmol of both drugs attenuated the reflex in adjuvant-treated and control animals indicating that effects of RP67580 at this dose were not mediated entirely by its action at NK1 receptors. Overall, these data suggest that endogenous substance P has a role in the increased excitability of spinal interneurons observed during persistent inflammation and support the hypothesis that substance P released in the spinal cord contributes to the hyperalgesia that accompanies adjuvant-induced persistent, peripheral inflammation.

Differential distribution of calbindin-D28k and parvalbumin in somatic and visceral sensory neurons.

The purpose of the present investigation was to determine whether calbindin-D28k and parvalbumin are distributed to different subpopulations of somatic and visceral sensory neurons. Immunofluorescent and retrograde techniques were combined to examine the distribution of calbindin- and parvalbumin-like immunoreactivity in the cell bodies of somatic and visceral primary afferent neurons in dorsal root ganglia L1-S1 of rats. Calbindin and parvalbumin were differentially distributed to essentially non-overlapping subpopulations of primary sensory neurons that could be distinguished by their segmental and size distributions, as well as by their innervation of somatic and visceral structures. Calbindin-like immunoreactivity was found in a population of smaller-sized cell bodies comprising approximately 14%of all dorsal root ganglion cells examined, with the proportions being greatest in L6 and S1. In contrast, parvalbumin was found in a population of larger-sized cells that made up about 11% of dorsal root ganglion cells and that were most concentrated in L4 and L5. Sensory neurons were further characterized by retrograde transport following the application of the neuroanatomical tracer FluoroGold to somatic (sural and gastrocnemius) and visceral (hypogastric and pelvic) nerves. Somatic tissues were innervated by a population of calbindin-containing as well as a separate population of parvalbumin-containing sensory neurons. In contrast, afferent neurons innervating visceral structures contained only a subpopulation of calbindin-containing neurons and very few parvalbumin-positive cells.

Immunohistochemical localization of delta- and mu-opioid receptors in primate spinal cord.

Immunofluorescent techniques were used to examine the distribution of delta- and mu-opioid receptor-like immunoreactivity (ir) in primate spinal cord. At every segmental level examined, opioid receptor-ir formed a dense plexus of small profiles within the superficial dorsal horn. delta-Opioid receptor-ir was found in laminae I and II, whereas mu-opioid receptor-ir was confined mostly to lamina II. The distribution of opioid receptors within the superficial dorsal horn is appropriate for a localization to the central terminals of thinly myelinated and unmyelinated sensory neurons. Differences in the distribution of the delta- and mu-opioid receptors may reflect the presence of these two opioid receptor types on different functional classes of afferent neurons, or they may relate to differences in presynaptic and postsynaptic localization within the spinal gray matter.

Distribution of four types of synapse on physiologically identified relay neurons in the ventral posterior thalamic nucleus of the cat.

This study was aimed at providing quantitative data on the thalamic circuitry that underlies the central processing of somatosensory information. Four physiologically identified thalamocortical relay neurons in the ventral posterior lateral nucleus (VPL) of the cat thalamus were injected with horseradish peroxidase and subjected to quantitative electron microscopy after pre- or postembedding immunostaining for gamma-aminobutyric acid to reveal synaptic terminals of thalamic inhibitory neurons. The four cells all had rapidly adapting responses to light mechanical stimuli applied to their receptive fields, which were situated on hairy or glabrous skin or related to a joint. Their dendritic architecture was typical of cells previously described as type I relay cells in VPL, and they lacked dendritic appendages. Terminals ending in synapses on the injected cells were categorized as RL (ascending afferent), F (inhibitory), PSD (presynaptic dendrite), and RS (mainly corticothalamic) types and were quantified in reconstructions of serial thin sections. RL and F terminals formed the majority of the synapses on proximal dendrites (approximately 50% each). The number of synapses formed by RL terminals declined on intermediate dendrites, but those formed by F terminals remained relatively high, declining to moderate levels (20-30%) on distal dendrites. RS terminals formed moderate numbers of the synapses on intermediate dendrites and the majority (> 60%) of the synapses on distal dendrites. Synapses formed by PSDs were concentrated on intermediate dendrites and were few in number (approximately 6%). They formed synaptic triads with F terminals and rarely with RL terminals. On somata, only a few synapses were found, all made by F terminals. The total number of synapses per cell was calculated to be 5,584-8,797, with a density of 0.6-0.9 per micrometer of dendritic length. Of the total, RL terminals constituted approximately 15%, F terminals approximately 35%, PSD terminals approximately 5%, and RS terminals approximately 50%. These results provide the first quantitative assessment of the synaptic architecture of thalamic somatic sensory relay neurons and show the basic organizational pattern exhibited by representatives of the physiological type of relay neurons most commonly encountered in the VPL nucleus.

Characteristics of intracellularly injected infragranular pyramidal neurons in cat primary auditory cortex.

Pyramidal neurons in layers V and VI of cat primary auditory cortex (AI) were intracellularly injected with biocytin after functional characterization according to a position relative to an anteroposterior sequence of best-frequency responses. A sample of 19 completely filled neurons was analyzed, and a preliminary classification was made on the basis of dendritic morphology and axon collateral distribution. Layer V cells could be divided into two types. Cells in the upper part of layer V and projecting toward the diencephalon had a large cell body and an apical dendrite with extensive branches in layer I. These cells had few recurrent axon collaterals, and no terminal axonal bushes were formed in the vicinity of the dendritic field. Long horizontal collaterals with many boutons, however, extended in various directions parallel to the cortical surface. By contrast, cells in the lower part of layer V and sending an axon into the putamen, or without an obvious subcortical axon, had a medium soma and an apical dendrite with few branches in layer I. These cells had a dense bush of recurrent collaterals extending into layers II and III and surrounding the dendritic field, but few or no horizontal collaterals. Layer VI injected neurons were more heterogeneous. All had a thin ascending dendrite with oblique branches both ending in layer III. Axon collateral distributions varied from cell to cell. Relatively small cells with an apical dendrite that branched frequently in layers III and IV had a dense network of recurrent collaterals in the dendritic field, but virtually no horizontal collaterals. This type projected toward the diencephalon. Cells with relatively long horizontal collaterals and a weak recurrent system confined to layers V and VI had a unique arborization pattern of basal dendrites. This type may have projected to the claustrum or other cortical areas. One cell with dendritic branches restricted to layer VI had horizontal collaterals predominantly in layer VI. This cell projected into the corpus callosum. The apparent close correlation between extrinsic projections of infragranular neurons and their dendritic morphology and intracortical collateral distributions suggests that differentially projecting cells may engage different elements of intracortical circuitry in AI.

Electrophysiological study of spinothalamic inputs to ventrolateral and adjacent thalamic nuclei of the cat.

1. Extracellular and intracellular methods were used to record from fibers and neurons in the ventral lateral (VL) and adjacent nuclei of the cat thalamus. The receptive fields of the recorded units were analyzed and the units tested for inputs from the medial lemniscus (ML) and spinothalamic tract (STT) by electrical stimulation of the dorsal columns (DC) and ventrolateral funiculus (VLF) at the C2-3 spinal level. 2. Thirty-eight STT fibers were isolated in the thalamus. Their conduction velocities ranged from 15 to 75 m/s (mode 36 m/s). Adequate stimuli were found for 23 of these fibers. Seventeen were low-threshold (LT), 3 were wide-dynamic-range (WDR), and 3 were high-threshold (HT) units. 3. Five STT fibers were intra-axonally injected. Three were sufficiently well filled for analysis of their terminal fields. An intermediate-velocity STT fiber (conduction velocity 38 m/s) had a 4.3-microns axon and a single large terminal field in the central lateral nucleus (CL). The other two STT fibers were smaller, with diameters of 2.5 and 2.3 microns, conduction velocities of 15 and 19 m/s, and terminal fields made up of a few small boutons at the borders of the ventral posterior lateral nucleus (VPL). 4. Of 319 neurons isolated, 14 out of 129 (10.8%) in VL, 14 out of 76 (18.4%) in the VPL or ventral posterior medial (VPM) nucleus, 27 out of 64 (42.2%) in the CL nucleus, and 5 out of 50 (10%) in the reticular nucleus (R) responded at latencies less than 50 ms to VLF stimuli. A train of three pulses was more effective in driving VLF-responding neurons in all these nuclei than a single pulse. VLF-responding cells were widely dispersed in VL, concentrated in a focus in CL, and distributed around the borders of VPL. Most of those in VL and a small number in CL could be antidromically activated by stimulation of motor cortex. 5. Latencies of presynaptic responses (STT fibers) to VLF stimulation were short and varied from 0.8 to 3.9 ms (mode 1.6 ms). Despite this, very few fast-responding neurons were found. These were six VPL neurons (2.5 to 4 ms), one VL neuron (3 ms), and four CL neurons (3-4 ms). The initial spike latencies of the majority of thalamic neurons responding to VLF stimulation appeared in two peaks, one between 6 and 8 ms and the other at 10-15 ms.(ABSTRACT TRUNCATED AT 400 WORDS)

Descending adrenergic input to the primate spinal cord and its possible role in modulation of spinothalamic cells.

The present study focuses on 3 different aspects of the descending adrenergic system in the primate: (1) the distribution of adrenergic fibers and terminals in the spinal cord, (2) the source of this input and (3) the possible physiological effects of this system on spinal nociceptive processing. Antibodies to the enzyme phenylethanolamine-N-methyltransferase (PNMT) were employed to map the distribution of epinephrine-containing axonal profiles in the primate spinal cord. Smooth longitudinally oriented fibers were localized to the outer edge of the lateral funiculus. PNMT-containing axonal enlargements were distributed to the superficial dorsal horn, intermediate gray matter and the region surrounding the central canal at all spinal cord levels. PNMT-immunostained profiles were also observed in the intermediolateral cell column. A double labeling study employing retrograde transport of HRP from the spinal cord and PNMT immunohistochemistry identified a small population of HRP-PNMT-labeled neurons in the 'C1' region at the levels of the medulla and ponto-medullary junction. Thus, these cells are a probable source of adrenergic input to the spinal cord. Electrophysiological studies demonstrated that iontophoresis of epinephrine onto identified primate spinothalamic tract neurons in the lumbar dorsal horn resulted in inhibition of the glutamate-induced firing of these cells. The data from these studies support the hypothesis that adrenergic (PNMT-containing) cells in the caudal brainstem project to all levels of the cord and may contribute to descending modulation of nociceptive processing at these levels.

Patterns of axon collateralization of identified supragranular pyramidal neurons in the cat auditory cortex.

Nine pyramidal neurons in layers II and III of cat primary auditory cortex (AI) were fully reconstructed after intracellular injections of horseradish peroxidase or biocytin. Each neuron was functionally characterized according to its position relative to an anteroposterior sequence of best frequency responses. All labeled somata were in layers II or III and gave rise to typical apical and basal dendritic arbors as well as to extensive systems of axon collaterals. The primary axon of all except 1 cell entered the white matter and was probably directed toward other cortical areas ipsi- or contralaterally. Two major intracortical collateral systems emerged from the main axon in AI, one ending in the vicinity of the cell and the second at a distance. (1) Many local and recurrent collaterals, given off in layers III and V, contributed terminal branches to the formation of a columnar pattern of terminations extending superficially and deeply into the soma. The column extended through layers I-V, with some constriction in the middle portion corresponding to layer IV. (2) The axon of each cell also gave rise to 2-5 thick, long-range collaterals in layers III and/or V. These ran parallel to the pial surface for several millimeters. At several points along these long horizontal collaterals, vertically directed branches emerged to form columnar terminations, again extending through layers I-V. These columns did not overlap with that formed in the vicinity of the cell, and were situated at distances 500-1200 microns from the cell body. When viewed in the tangential plane, horizontal collaterals were oriented, on the whole, dorsoventrally with respect to the surface of the cortex. This may correspond to the organization of isofrequency bands previously described in cats. The results suggest that the major spread of excitation in AI is mediated by horizontal collaterals of pyramidal cells and that it occurs along the lines of isofrequency domains. Within the latter the collaterals may link columns of cells with like properties and/or serve to coordinate activity patterns in spatially separated portions of AI.

Distribution of phenylethanolamine N-methyltransferase cell bodies, axons, and terminals in monkey brainstem: an immunohistochemical mapping study.

Adrenaline (epinephrine) is an important candidate transmitter in descending spinal control systems. To date intrinsic spinal adrenergic neurons have not been reported; thus adrenergic input is presumably derived from brainstem sites. In this regard, the localization of adrenergic neurons in the brainstem is an important consideration. Maps of adrenergic cell bodies and to a lesser extent axons and terminal fields have been made in various species, but not in monkeys. Thus, the present study concerns the organization of adrenergic systems in the brainstem of a monkey (Macaca fascicularis) immunohistochemically mapped by means of an antibody to the enzyme phenylethanolamine N-methyltransferase (PNMT). PNMT-immunostained cell bodies are distributed throughout the medulla in two principal locations. One concentration of labeled cells is in the dorsomedial medulla and includes the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (X), and an area ventral to X in a region of the reticular formation (RF) known as the central nucleus dorsalis (CnD) of the medulla. A few scattered cells are observed in the periventricular gray just ventral to the IVth ventricle and on midline in the raphe. The second major concentration of PNMT-immunostained cells is located in the ventrolateral RF, lateral and dorsolateral to the inferior olive (IO), including some cells in the rostral part of the lateral reticular nucleus (LRN). Terminal fields are located in the NTS, X, area postrema (AP), and the floor of the IVth ventricle in the medulla and pons. A light terminal field is also observed in the raphe, particularly raphe pallidus (RP). A heavy terminal field is present in locus coeruleus (LC). Fibers labeled for PNMT form two major fiber tracts. One is in the dorsomedial RF extending as a well-organized bundle through the medulla, pons, and midbrain. A second tract is located on the ventrolateral edge of the medulla and caudal pons. Fibers in this tract appear to descend to the spinal cord. A comparison with maps of other catecholamine neurons in primates is discussed, confirming that the distribution of the adrenergic system in monkeys is similar to that described in the human.

Ultrastructural analysis of axosomatic contacts on functionally identified primate spinothalamic tract neurons.

The morphology and frequency of axosomatic contacts on three functionally identified primate spinothalamic tract (STT) cells were analyzed at the electron microscopic level. The STT cells analyzed were wide-dynamic-range neurons responsive to activation of low- and high-threshold cutaneous afferents innervating the foot. The somas were located in the lateral border of lamina V; the dendritic trees were oriented dorsally and were very extensive. Numerous spinelike appendages were observed emanating from two of the cell bodies. Terminal types contacting the cell bodies were categorized at several different layers through each neuron. Six morphologically different terminal types were established following analysis of serial sections. Profiles classified as round (R) terminals containing round clear vesicles and zero or one dense-core vesicle made up over 50% of the total population in contact with the STT somas. Profiles containing round clear vesicles and two to four small-diameter dense-core vesicles (D1 category) made up approximately 10% of the population in contact with each soma. Flat (F) terminals with oblong or flattened clear vesicles made up approximately 8% of the population. The remaining three categories (D2, L1, and L2) distinguished by the number and size of the dense-core vesicles made up a small percentage of the total population in contact with the cell bodies. The distribution of terminal types on the soma proper versus somatic spines was also determined for one cell. The proportions of the six terminal types contacting the soma of these cells were very similar, although the physiological characteristics of each cell were different. However, the relative proportions of terminal types on these three lamina V STT cell bodies were different from those previously reported contacting somata in lamina V, suggesting that there may be a unique innervation of STT cells that differentiates them from other cell types in lamina V.

Patterns of spontaneous discharge in primate spinothalamic neurons.

1. The spontaneous discharge of 30 spinothalamic tract (STT) neurons in the lumbosacral spinal cord of anesthetized monkeys was studied. Interval, correlation, and spectral analyses were performed. 2. Three patterns of discharge were found; these were referred to as the SP1, SP2, and SP3 patterns. 3. The SP1 group had moderately regular discharge trains that were devoid of short-interval spike bursts. 4. The SP2 group had spike trains dominated by short-interval bursts without evidence of low-frequency rhythmicity. 5. The SP3 group had spike trains with features of both the SP1 and SP2 groups. 6. Some correlations were found between the mean discharge rate and stimulus-response classes previously defined by our group (25). Correlations were deduced from a parent data set of 221 STT neurons. Type 1 neurons, which were driven primarily by tactile afferents, were found to have significantly lower mean rates than other "within-neuron" groups. On the other hand, type C neurons, which had strong input from afferents signalling pressure and noxious stimuli, were found to have significantly higher mean rates than all other "across-neuron" classes. 7. A weak relationship was found between the pattern of discharge and the within-neuron stimulus-response classification. Neurons with a largely tactile coding orientation (types 1 and 2) were most frequently of the SP1 and SP2 classes, whereas neurons with a more prominent nociceptive input (types 3 and 4) were most frequently of the SP2 and SP3 classes. No relationship was apparent between discharge pattern and the across-neuron classes.