Axons of sacral preganglionic neurons in the cat: II. Axon collaterals

Axon collaterals were identified in 21 of 24 preganglionic neurons in the lateral band of the sacral parasympathetic nucleus of the cat. Following the intracellular injection of HRP or neurobiotin the axons from 20 of these neurons were followed and 53 primary axon collaterals were found to originate from unmyelinated segments and from nodes of Ranvier. Detailed mapping done in the five best labeled cells showed bilateral axon collaterals distributions up to 25,000 microm in length with 950 varicosities and unilateral distributions up to 12,561 microm with 491 varicosities. The axon collaterals appeared to be unmyelinated, which was confirmed at EM, and were small in diameter (average 0.3 microm). Varicosities were located mostly in laminae I, V, VII, VIII and X and in the lateral funiculi. Most varicosities were not in contact with visible structures but some were seen in close apposition to Nissl stained somata and proximal dendrites. Varicosities had average minor diameters of 1.3 microm and major diameters of 2.3 microm. Most were boutons en passant while 10-20% were boutons termineaux. EM revealed axodendritic and axoaxonic synapses formed by varicosities and by the axons between varicosities. It is estimated that the most extensive of these axon collaterals systems may contact over 200 spinal neurons in multiple locations. These data lead to the conclusion that sacral preganglionic neurons have multiple functions within the spinal cord in addition to serving their target organ. As most preganglionic neurons in this location innervate the urinary bladder, it is possible that bladder preganglionic neurons have multiple functions.     

Location of bladder preganglionic neurons within the sacral parasympathetic nucleus of the cat.

Sacral parasympathetic preganglionic neurons innervating the urinary bladder were labelled by applying horseradish peroxidase (HRP) to branches of the pelvic nerve at the neck of the bladder. Bladder preganglionic cells were located primarily in the intermediolateral grey matter of sacral cord segments 1, 2 and 3 and extended from the ventral end of the central canal to the inferior margin of the dorsal horn. Dendrites extended into the dorsolateral funiculus, toward the lateral edge of the dorsal horn, medially beneath the dorsal horn and ventrally within the nucleus. Although a small number of cells were found medially beneath the dorsal horn, it is concluded that the majority of bladder preganglionic neurons occupy a lateral position within the sacral parasympathetic nucleus.     

Spinoreticular neurons in the second sacral segment of the feline spinal cord.

In continuation of previous electrophysiological studies on the location of ascending tract neurones within the second sacral segment of the feline spinal cord, the spinoreticular projections of these neurones have been investigated. Following electrical stimulation of the axonal terminals of 37 spinoreticular neurons via a tungsten electrode placed stereotactically in the contralateral nucleus reticularis gigantocellularis, antidromic potentials from their cell bodies were recorded with glass microelectrodes both extra- and intracellularly. The axons of these neurones were additionally excited from the dorsolateral funiculi of the contralateral (n = 37) and ipsilateral (n = 30) side at the lowermost thoracic spinal level. The latencies of antidromic excitation from the brainstem to the second sacral segment ranged from 3.2 to 11.8 ms (mean, 5.9 ms), whereas the corresponding axonal conduction velocities were between 27.1 and 100 m/s. The neurones examined in this study were found to be situated in the medial lamina VII of Rexed and the area adjacent to the central canal (n = 13), the medial lamina VIII (n = 12), medial laminae V and VI (n = 10) and in laminae II and III (n = 2). Three medium-sized (40-60 microm) of triangular- or oval-shaped neurones were visualized in medial laminae VII and VIII following the intracellular labelling with horseradish peroxidase.     

Electrophysiological properties of sympathetic preganglionic neurons in the cat spinal cord in vitro

Intracellular recordings were obtained from sympathetic preganglionic neurons of the intermedio-lateral nucleus of the adult cat in slices of upper thoracic spinal cord maintained in vitro. The neurons were identified by their antidromic responses to stimulation of various ipsilateral sites. Sites from which antidromic responses could be evoked were the white ramus, the ventral root, the ventral root exit zone, the white matter between the latter and the outer edge of the tip of the ventral horn, the lateral edge of the ventral horn. Resting membrane potential was –61.3±1.6 mV (mean±SEM), input resistance 67.5±3.7 M, time constant 11.5±1.2 ms. The amplitude of the action potential generated by antidromic or direct stimulation was 77.4±2.3 mV. Threshold for direct spikes was 18.2±1.8 mV. The action potential had an average duration of 3.03±0.16 ms. It showed a prominent hump on the falling phase. The action potential had a tetrodotoxin (TTX)-sensitive and a TTX-resistant component. The latter was abolished by cobalt.Tetraethylammonium, cesium and barium prolonged the action potential duration which acquired a plateau-shape. A prolonged after-hyperpolarization (AHP) followed the sympathetic preganglionic neuron spike. Following a single spike, AHP duration and peak amplitude were 2.8±0.3 s and 16.6±0.7 mV, respectively. The AHP was abolished by cesium or barium, but enhanced by tetraethylammonium. An AHP followed the TTX-resistant spike. EPSPs and IPSPs could be generated by focal stimulation. The EPSP triggered spikes when threshold (15.0±2.0 mV) was reached. The slice of the thoracic spinal cord provides a useful experimental preparation for analysis of cellular properties and synaptic mechanisms of the sympathetic preganglionic neuron.     

In vivo assembly of the axon initial segment in motor neurons

The axon initial segment (AIS) is responsible for both the modulation of action potentials and the maintenance of neuronal polarity. Yet, the molecular mechanisms controlling its assembly are incompletely understood. Our study in single electroporated motor neurons in mouse embryos revealed that AnkyrinG (AnkG), the AIS master organizer, is undetectable in bipolar migrating motor neurons, but is already expressed at the beginning of axonogenesis at E9.5 and initially distributed homogeneously along the entire growing axon. Then, from E11.5, a stage when AnkG is already apposed to the membrane, as observed by electron microscopy, the protein progressively becomes restricted to the proximal axon. Analysis on the global motor neurons population indicated that Neurofascin follows an identical spatio-temporal distribution, whereas sodium channels and β4-spectrin only appear along AnkG⁺ segments at E11.5. Early patch-clamp recordings of individual motor neurons indicated that at E12.5 these nascent AISs are already able to generate spikes. Using knock-out mice, we demonstrated that neither β4-spectrin nor Neurofascin control the distal-to-proximal restriction of AnkG.     

Relation between axons and oligodendroglial cells during initial myelination I. The glial unit

Summary The morphology of oligodendroglial-axon units was examined by electron microscopy during ensheathment and initial myelination in developing feline spinal cord and corpus callosum white matter. In addition to a qualitative examination of single sections from many stages of development, a morphological analysis of spinal cord and corpus callosum units was made on the basis of serial sections from a few stages. The results show that myelination commences around embryonic/fetal day 40 and the 20th postnatal day in the spinal cord and corpus callosum areas, respectively. In both areas immature glial cells, lacking the cytological features of typical oligodendrocytes, initially associate with several axons and provide them with cytoplasmic sheaths. Serial section analysis of units, which have begun formation of compact myelin, indicates that individual cells are associated with single myelin sheaths in the spinal cord area, in a way principally similar to the Schwann cell-myelin units in developing peripheral nerves. This suggests the possibility that early spinal cord oligodendrocytes might shift from a polyaxonal to a monoaxonal association after initial ensheathment and before formation of compact myelin. In the corpus callosum area the examined serially-sectioned cells were found to be connected to several myelin sheaths through long thin processes. The myelin sheaths related to one cell are relatively uniform in terms of number of myelin lamellae and axon diameter, but the clockwise/counter-clockwise course of the myelin spiral varies randomly. Units containing both homogeneously uncompacted (cytoplasmic) and fully compacted (myelin) sheaths have not been found. In both areas the ensheathing cells achieve an oligodendrocyte-like cytology during formation of the first layers of compact myelin. These observations support the view that oligodendrocytes are structurally heterogeneous: those myelinating prospective large axons seem to differ from those myelinating axons destined to remain small. The possible functional and pathophysiological implications of this heterogeneity remain to be elucidated.     

Somatosensory response properties of contralaterally projecting spinothalamic and nonspinothalamic neurons in the second cervical segment of the cat.

1. The upper cervical spinal cord contains over one-third of the cells of the spinothalamic tract (STT). This study investigated response properties of contralaterally projecting STT neurons in C2 of the cat by the use of single-unit, microelectrode recordings. Standard antidromic stimulation and collision techniques were used to identify STT units projecting to the contralateral thalamus. Once an STT unit was found, its receptive field (RF) and responses to cutaneous stimuli such as touch, pressure, deep muscle squeeze, tap, noxious pinch, and heat were characterized. C2 units that were not activated from the contralateral thalamus (non-STT units) were also characterized. The locations of thalamic stimulation electrodes and spinal recording sites were reconstructed from electrolytic lesions. 2. A total of 48 STT and 68 non-STT units were well characterized. RF sizes were classified as small, intermediate, large, or whole body. Each unit was also classified as having one of two possible response types: simple units were those with homogeneous responses within the RF and were classified as low threshold (LT), high threshold (HT), wide dynamic range (WDR), deep, or tap. Complex units were those that responded differently in different regions of the RF. 3. The average depth of non-STT units subdivided by RF size was 2.1 +/- 0.6 (SD) mm for cells with small RFs, 2.4 +/- 0.8 mm for cells with intermediate RFs, 2.8 +/- 0.3 mm for cells with large RFs, and 2.7 +/- 0.5 mm for cells with whole-body RFs. The average depth of non-STT units based on response type was 2.0 +/- 0.5 mm for LT, 2.3 +/- 0.7 mm for HT, 2.1 +/- 0.7 mm for WDR, 2.6 +/- 0.9 mm for deep, 2.6 +/- 0.5 mm for tap, and 2.4 +/- 0.2 mm for complex. 4. A somatotopic organization along the rostrocaudal length of C2 and upper C3 was observed for non-STT units with small- and intermediate-size RFs. The average distance of the recording sites from the rostralmost dorsal rootlet of C2 was 3.8 +/- 2.1 mm for units with RFs on the face, 7.1 +/- 4.3 mm for units with RFs on the neck, and 11.9 +/- 5.1 mm for units with RFs on the forelimb. 5. The average threshold for antidromic activation of STT units was 175 +/- 120 microA. Most C2 STT units were activated from the ventroposterior region of the thalamus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Afterdepolarization mechanism in the in vitro, cesium-loaded, sympathetic preganglionic neuron of the cat

Intracellular recordings were performed in Cs-loaded sympathetic preganglionic neurons (SPNs) of the intermediolateral nucleus, identified by antidromic stimulation, in the slice of the T2 or T3 segment of the cat spinal cord. Loading the neurons with Cs resulted in broadening of the action potential, depression of the fast component of the afterhyperpolarization (AHP), and appearance of an afterdepolarization (ADP). A typical ADP in a Cs-loaded neuron had time to peak of 45-110 ms, half-decay time of 70-250 ms, and amplitude of 2-10 mV at membrane potentials between -60 and -70 mV and at a Ca and K concentration of 2.5 and 3.6 mM, respectively, in the superfusion medium. The ADP was associated with a decrease in neuron input resistance and increased in magnitude with hyperpolarization of the cell membrane. The relation between peak ADP amplitude and membrane potential was linear within the range of membrane potentials from -60 to -100 mV. The ADP was reversibly suppressed by the Ca-channel blocker cobalt (2 mM) or by low Ca Krebs solution (0.25 mM). Superfusion with BaCl2 (1.0 mM) or tetraethylammonium (TEA) (10-20 mM) caused an increase in amplitude of the ADP and an increase in action potential duration. Hyperpolarizing pulses, delivered during the course of the spike shoulder, resulted in a decrease of spike duration and ADP amplitude. The ADP was not affected by tetrodotoxin, at a dose blocking the Na-spike, and was enhanced, in association with an increase in action potential duration, when NaCl in the Krebs solution was replaced with choline chloride. Increasing intracellular Cl concentration or decreasing extracellular Cl concentration had no effect on the ADP. Changes in external K concentration from 3.6 to 10 or 0.36 mM increased and decreased, respectively, the amplitude of the ADP. In the absence of Cs, and ADP, with similar time course to that recorded in Cs-loaded SPNs, was recorded when CaCl2 was replaced by BaCl or NaCl was replaced by TEAC1. It is concluded that the SPN afterpotential includes a Ca-dependent inward current, in addition to the already described fast and slow outward K currents of the AHP.     

Fast excitatory postsynaptic potentials and the responses to excitant amino acids of sympathetic preganglionic neurons in the slice of the cat spinal cord.

The properties of the excitatory postsynaptic potential evoked by focal stimulation and of the responses to excitatory amino acids were examined by intracellular recording from sympathetic preganglionic neurons in upper thoracic spinal cord slices of the adult cat. Single stimuli to the region dorsal to the intermedio-lateral nucleus evoked short-latency, presumably monosynaptic, excitatory postsynaptic potentials. The reversal potential of this response was -2.2 mV and became more negative when external Na+ or K+ concentration was decreased. The excitatory postsynaptic potential was depressed by the non-selective excitatory amino acid receptor antagonist cis-2,3-piperidine dicarboxylic acid and enhanced by a glutamate uptake inhibitor. The non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2.3-dione abolished the excitatory postsynaptic potential in 72% of neurons. In the remaining neurons, this antagonist only depressed the potential and unmasked a slower component which was abolished by the N-methyl-D-aspartate receptor antagonist D,L-2-amino-5-phosphonovaleric acid. In the presence of tetrodotoxin all neurons tested were depolarized by glutamate or aspartate, as well as by the selective agonists quisqualate, alpha-amino-3-hydroxy-5-methylisoxazole propionic acid, kainate and N-methyl-D-aspartate. The glutamate-evoked depolarization reversed at a membrane potential of -2.0 mV and at a more negative value when external Na+ or K+ concentration was decreased. The response to alpha-amino-3-hydroxy-5-methylisoxazole propionic acid was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione in all neurons tested and that to kainate in only one-third of the cells. In the remainder the response to kainate was only slightly depressed by this antagonist. The responses to glutamate and aspartate were only slightly depressed by the combined action of the various amino acid receptor antagonists used. The responses to N-methyl-D-aspartate were abolished by D,L-2-amino-5-phosphonovaleric acid. The punched-out region of the intermedio-lateral nucleus, maintained in vitro, released glutamate and aspartate in the absence of stimulation. Field stimulation (20 Hz) enhanced release by between 40 and 100%. The increase was prevented by superfusion with calcium-free Krebs. It is concluded that excitatory amino acids, acting on both N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors, but mainly on the latter, are likely mediators of the monosynaptic excitatory postsynaptic potential evoked in sympathetic preganglionic neurons by the stimulated region. The efflux data suggest that glutamate and aspartate are among the mediators.     

The dorsal motor nucleus of the vagus nerve of the cat: Localization of preganglionic neurons by quantitative histological methods

The dorsal motor nucleus of the vagus nerve (DMX) of adult cats and young kittens was studied by quantitative light microscopic methods. In normal animals, the DMX was found to contain no distinct subgroupings of neurons, based on somatic volume or Nissl pattern. Retrograde perikaryal responses to axotomy of neurons in the DMX were found to be of a more subtle nature than those seen in other types of neurons. Quantitative methodology applied to the DMX after cervical vagotomy permitted a better understanding of the results of axotomy than could be obtained by routine microscopic observations. Changes which occurred included a slight chromatolytic reaction, and a decrease in the volume of the nucleus followed by an increase in somatic volume. These morphological alterations were affected by the factors of age of the animal, time after axotomy, and length of the intact proximal axon stump. More pronounced perikaryal changes occurred when the vagus nerve was recut at a more proximal level five days after the first vagotomy. Interpretation of the data yielded the conclusion that most if not all neurons of the ipsilateral DMX contribute axons to the cervical vagus nerge. In addition, at least 10% of the neurons on the side contralateral to vagotomy showed signs of retrograde reaction. It was therefore concluded that there exists in the vagus nerve a population of axons with cell bodies located in the contralateral DMX.     

Origin,course and termination of dopaminergic substantia nigra neurons projecting to the amygdaloid complex in the cat

The organization of dopaminergic neurons projecting to the amygdala was examined using retrograde (horseradish peroxidase histochemistry) and anterograde ([³H]leucine autoradiography) transport methods and Falck-Hillarp histofluorescence techniques combined with microspectrofluorometry and radiofrequency lesions. Cell bodies located within the pars lateralis and pars compacta of the substantia nigra were found to project to the lateral and central amygdaloid nuclei, respectively. Both of these areas within the substantia nigra contained dopaminergic perikarya, while the central and lateral amygdaloid nuclei contained fluorescent varicosities with features indicative of dopaminergic neurons. Lesions restricted to the pars lateralis of the substantia nigra resulted in a loss of fluorescence in the lateral amygdaloid nucleus. Autoradiographic experiments revealed that the projections from the pars lateralis did not run with fibers originating from the pars compacta in the nigrostriatal tract but rather had their own course occupying a lateral position adjacent to the cerebral peduncle and joining the ventral amygdalo-fugal bundle.The data indicate that, in the cat, there are two separate dopaminergic projections to the amygdala arising from the substantia nigra.     

The location of cardiac vagal preganglionic motoneurones in the medulla of the cat.

1. Electrophysiological techniques have been used to locate the origin of preganglionic vagal motoneurones supplying the heart of the cat. 2. The right cardiac vagal branches were identified anatomically and their ability to slow the heart was assessed by electrical stimulation. Control experiments revealed that contamination of cardiac branches by bronchomotor and oesophageal efferent fibres was likely to be small. 3. Fifty-seven neurones in the medulla were activated antidromically on stimulating the cardiac branches at up to 5 times the threshold for cardiac slowing. They had axons with conduction velocities between 3 and 15 m/sec, corresponding to B fibres. 4. None of these were located in the region of the dorsal motor nucleus of the vagus, in spite of repeated sampling there, but all were located in the region of the nucleus ambigus. Histological examination of marked neurones (forty-six of the fifty-seven neurones) revealed that they were associated with its principal column, rostral to the obex. 5. Sampling motoneurones of the dorsal motor nucleus revealed that most sent axons down the thoracic vagus below the cardiac branches. Only three of thirty-three could be activated antidromically by high intensity stimulation of the cardiac branches, but on the basis of their thresholds and conduction velocities, it is argued that they were unlikely to be cardio-inhibitory neurones. 6. It is concluded that preganglionic cardio-inhibitory neurones arise not in the dorsal motor nucleus, but in the principal column of the nucleus ambiguus.     

Visceral and somatic afferent convergence onto neurons near the central canal in the sacral spinal cord of the cat

One hundred and sixty extracellularly and intracellularly recorded unitary discharges from the sacral or caudal spinal segments of 30 anemically decerebrated cats were studied to examine the effects of somatic and visceral afferent stimulation on neurons near the central canal (CC). The recorded unitary activity was histologically verified (by dye marks or horseradish peroxidase, HRP) as having come from the gray matter surrounding the CC that approximates Rexed's lamina X. In the absence of intentional stimulation or apparent injury by the recording electrode, 62% of the units exhibited ongoing discharges. Each unit was tested for responses to the stimulation of somatic (cutaneous and subcutaneous) and visceral (bladder and colon) structures. Seventy-six (48%) of the units responded exclusively to the stimulation of somatic receptive fields, and 10 (6%) of the units were selectively responsive to stimulation of the pelvic viscera. The activity of the remaining 74 (46%) was influenced by activity in both somatic and visceral afferent fibers. Eighteen of the 160 neurons were intracellularly marked with HRP. Based on perikaryal size and dendritic extent, it was possible to divide these cells into two partially overlapping groups. One group consisted of seven neurons with small to medium-sized perikarya, dendritic arbors largely restricted to the gray matter surrounding the CC, and small, singular somatic receptive fields. The second group comprised 11 cells with medium to large-sized soma and dendrites extending out of lamina X. These larger neurons usually possessed multiple, widely distributed somatic receptive fields. The principal finding of the present study is that in the sacral spinal cord many cells near the CC receive primary afferent inputs converging from a wide range of receptor types in somatic and visceral structures. Such neurons are capable of integrating afferent information from somatic structures on both sides of the body with information originating in pelvic viscera and midline regions such as the genitals.     

NADPH diaphorase-positive neurons in the rat spinal cord include a subpopulation of autonomic preganglionic neurons.

Preganglionic neurons in the spinal cord of the rat were labelled retrogradely with Fluoro-gold and the spinal cord stained for NADPH diaphorase. The majority of both sympathetic and sacral parasympathetic preganglionic neurons showed staining for NADPH diaphorase. NADPH diaphorase-positive neurons were located more laterally in the intermediate zone than were preganglionic neurons lacking NADPH diaphorase staining. The recent evidence that identifies NADPH diaphorase as nitric oxide synthase raises the possibility that some spinal preganglionic neurons may synthesize nitric oxide.     

Ultrastructure and synaptology of the initial axon segment of cat spinal motoneurons during early postnatal development

Summary The initial axon segment (IS) of spinal -motoneurons has been studied in single and serial sections during the first three postnatal weeks in the cat. It was found that boutons of different ultrastructural types made synaptic contact with the IS during the first postnatal week but during the second postnatal week the boutons disappeared. The diameter of the IS was not observed to change from the first to the second postnatal week but a significant increase was noted from the second to the third week. The length and general ultrastructural appearance of the IS did not change significantly over the postnatal period studied. The results are discussed in relation to some aspects of the functional development of spinal motoneurons during the early postnatal period.