Morphology of sympathetic preganglionic neurons in the thoracic spinal cord of the cat: an intracellular horseradish peroxidase study.

Horseradish peroxidase was intracellularly injected into sympathetic preganglionic neurons (SPN) of the third thoracic segment in cats. Seven neurons were reconstructed from serial horizontal or parasagittal sections of the spinal cord. The cell bodies of all neurons were located in the n. intermediolateralis pars principalis (ILp). They were spindle-shaped with the long axis in craniocaudal direction or large and multipolar or small and oval in shape. Preferentially on the cranial and caudal pole of the cell body, five to eight primary dendrites arose from the cell body. Dendritic branches were traced to their terminations at distances up to 1,330 microns from the cell body. The dendritic fields of all SPNs were strictly oriented in the longitudinal direction with a total length of 1,500-2,540 microns. The cranial and caudal dendritic fields were about equal in length but, with one exception, the degree of branching was always greater in the cranial than in the caudal dendritic field. The dendritic fields of all SPNs were primarily restricted to the ILp. In the mediolateral direction it extended from 130 to 360 microns and in the dorsoventral direction from 50 to 180 microns. Only rarely, a higher-order dendrite left the boundaries of the ILp and projected dorsolaterally or laterally into the white matter or ventromedially or medially into the adjacent n. intercalatus. All dendrites showed various forms of spines. At a distance of 132-437 microns from the cell body the axon arose as a direct extension of a process which closely resembled a primary or second-order dendrite. The axons projected ventrally and mostly caudally along the lateral border of the gray matter until they turned laterally at the end of the ventral horn. No axon collaterals were observed.

Rhythmic phrenic, intercostal and sympathetic activity in relation to limb and trunk motor activity in spinal cats.

During L-DOPA-induced fictive spinal locomotion rhythmic activities in nerves to internal intercostal and external oblique abdominal muscles and in phrenic and sympathetic nerves were observed which were always coordinated with locomotor activity in forelimb and hindlimb muscle nerves. A periodicity with longer lasting tonic phases could be induced by cutaneous nerve stimulation or asphyxia. This activity was observed in limb motor nerves as well as in respiratory motor and sympathetic nerves. A slow independent activity of the phrenic and intercostal nerves or the sympathetic nerves, which could be related to a normal respiratory rhythm or independent sympathetic rhythms was not observed. The findings indicate that during fictive spinal locomotion the activity of spinal rhythm generators for locomotion also projects onto respiratory and sympathetic spinal neurones.

Rostrocaudal location of sympathetic preganglionic neurones within the third thoracic segment of the cat spinal cord investigated by the retrograde transport of horseradish peroxidase and by recording of antidromic field potentials.

The rostrocaudal location of sympathetic preganglionic neurones (SPNs) in the intermediolateral cell column of the third thoracic segment was studied in the cat by the retrograde transport of horseradish peroxidase and by recording of antidromic field potentials in the spinal cord in response to stimulation of white ramus T3. By both methods, the position of the rostral and caudal border of SPNs was determined in relation to the entry of segmental dorsal roots. It was found that SPN's are confined in the spinal cord to the length of one segment (9494 +/- 823 micron), but are shifted rostrally by about 3 mm with respect to the point of entry of the dorsal roots of segment T3.

Baroreceptor inhibition of subretrofacial neurons: evidence from intracellular recordings in the cat.

Neurons of the subretrofacial nucleus in the rostral ventrolateral medulla have been studied by in vivo intracellular recording in the chloralose-anaesthetized cat. Impalements of sufficient quality to demonstrate inhibition by carotid baroreceptor stimulation (blind sac inflation) were obtained for 9 cells. In 8 of these, a clear hyperpolarization followed approximately 100-200 ms after the baroreceptor stimulus, reaching a maximum of 2-9 mV, 200-500 ms later. These findings confirm by more direct methods the inhibition of subretrofacial neurons by arterial baroreceptors.

Inhibition of cardiac sympathetic nerve activity during swallowing evoked by laryngeal afferent stimulation in the cat.

Spontaneous swallowing or the buccopharyngeal phase of swallowing evoked by electrical stimulation of the superior laryngeal nerve was accompanied by a pronounced decrease of sympathetic activity in the cardiac nerve. This reduction of sympathetic activity was not related to other influences such as postinspiratory inhibition or baroreceptor-mediated inhibition. Intracellular recordings from sympathetic preganglionic neurones revealed hyperpolarization during the buccopharyngeal phase of swallowing, possibly due to postsynaptic inhibition.

Segmental distribution of afferent fibres in the left inferior cardiac nerve of the cat studied by anterograde transport of horseradish peroxidase.

The segmental projection of afferent fibres in the left inferior cardiac nerve of the cat was studied by using the anterograde transport of horseradish peroxidase (HRP). HRP-positive cell bodies were detected in ipsilateral dorsal root ganglia from segments T1 to T7. No labelled neurones were found at segments C8 and T8. The total number of labelled neurones ranged from 157 to 535 neurones in individual experiments. The majority of neurones (70-88%) were localized in the dorsal root ganglia of segments T2-T4. Labelled neurones were oval shaped, and most neurones had a long axis in the range from 20 to 40 microns.

Morphology of electrophysiologically identified baroreceptor afferents and second order neurones in the brainstem of the cat.

Baroreceptor afferent fibres and second order baroreceptor neurones were identified by their discharge pattern and were intracellularly injected with horseradish peroxidase. Three afferent fibres and three second order neurones were reconstructed by camera lucida drawings from serial sections of the brainstem. The afferent fibres were classified as A delta-fibres and had terminal arborizations with synaptic boutons in the dorsomedial region of the nuclei of the solitary tract (TS). The afferent fibres had additional collaterals with a medial projection to the commissural nucleus and in a direction lateral to the TS. The terminals of these collaterals could not be demonstrated. The second order neurones were located in the same dorsomedial region as the synaptic boutons of the afferent fibres. Neurones were small and spindle-shaped with two primary dendrites: one dendrite projected cranially along the medial border of the TS, and the second one projected caudally and medially into the commissural nucleus. The unmyalinated axons of these neurones could be traced over a distance of 1 mm. In only one neurone could an axon collateral be detected. The axons projected dorsally around the TS in a ventrolateral direction beyond the boundaries of the nuclei of the TS. The axon collateral projected in the medial direction into the commissural nucleus. In no case were axon terminals demonstrated.

The novel CXCR4 antagonist POL5551 mobilizes hematopoietic stem and progenitor cells with greater efficiency than Plerixafor.

Mobilized blood has supplanted bone marrow (BM) as the primary source of hematopoietic stem cells for autologous and allogeneic stem cell transplantation. Pharmacologically enforced egress of hematopoietic stem cells from BM, or mobilization, has been achieved by directly or indirectly targeting the CXCL12/CXCR4 axis. Shortcomings of the standard mobilizing agent, granulocyte colony-stimulating factor (G-CSF), administered alone or in combination with the only approved CXCR4 antagonist, Plerixafor, continue to fuel the quest for new mobilizing agents. Using Protein Epitope Mimetics technology, a novel peptidic CXCR4 antagonist, POL5551, was developed. In vitro data presented herein indicate high affinity to and specificity for CXCR4. POL5551 exhibited rapid mobilization kinetics and unprecedented efficiency in C57BL/6 mice, exceeding that of Plerixafor and at higher doses also of G-CSF. POL5551-mobilized stem cells demonstrated adequate transplantation properties. In contrast to G-CSF, POL5551 did not induce major morphological changes in the BM of mice. Moreover, we provide evidence of direct POL5551 binding to hematopoietic stem and progenitor cells (HSPCs) in vivo, strengthening the hypothesis that CXCR4 antagonists mediate mobilization by direct targeting of HSPCs. In summary, POL5551 is a potent mobilizing agent for HSPCs in mice with promising therapeutic potential if these data can be corroborated in humans.

Integrative properties of sympathetic preganglionic neurones within the thoracic spinal cord.

The discharge pattern of sympathetic preganglionic neurones (SPNs) in the lateral horn is shaped by the interplay of synaptic inputs, membrane properties and local factors within the spinal cord. Intracellular recordings in vivo and in vitro have clarified the importance of some of these factors. Pacemaker activity can be recorded in vitro, but does not contribute to the generation of action potentials in vivo where spikes are solely generated from synaptic potentials. Synaptic potentials occur in phase with either the cardiac or the respiratory cycle or at irregular intervals. Postsynaptic interaction of these various inputs at the level of SPNs as well as presynaptic gating mechanisms in relation to the respiratory cycle have been observed. The discharge pattern is also modified by specific membrane properties which function to limit their discharge rate in the absence of axon collaterals. Finally the discharge of SPNs is affected by local factors: Since asphyxia causes a strong sympathetic activation when synaptic inputs to other neurones are already non-functioning synapses on SPNs are resistant to hypoxia or changes in the extracellular fluid somehow influence the activity of these neurones.

Three types of sympathetic preganglionic neurones with different electrophysiological properties are identified by intracellular recordings in the cat.

Intracellular recordings were obtained from sympathetic preganglionic neurones (SPN) of the third thoracic segment in cats. Based on differences in their active and passive electrophysiological properties, three different types of SPNs were discerned: Type A neurones had a high resting membrane potential (RMP) (-60 to -86 mV) and a low input resistance (RN) 12-23 M omega). Action potentials of these neurones had a pronounced IS-SD inflexion and a prominent shoulder in their falling phase. Spikes were rarely generated from the on-going synaptic activity. Type B neurones had a lower RMP (-48 to -65 mV) and a higher RN (21-37 M omega). Their action potentials were characterized by an after-depolarization; they showed a slight IS-SD inflexion and a less pronounced shoulder in their falling phase. The after-depolarization was abolished by membrane hyperpolarization in a time dependent way. A hyperpolarization of at least 50 ms duration was required for its abolition. The after-depolarization was also abolished during repetitive discharges. In most of these neurones spikes were generated at irregular intervals and low rates (0.06-4.6 spikes/s) from the synaptic activity. Type C neurones were similar to type B neurones, but their action potentials did not show the after-depolarization. Additionally, spikes were generated at fairly regular intervals and rather high rates (0.8-6.5 spikes/s). The rate of spike repolarization of all neurones was markedly increased by hyperpolarization and decreased by membrane depolarization. Current-voltage curves of some type B and C neurones showed a marked rectification upon membrane hyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of sympathetic reflexes evoked by electrical stimulation of phrenic nerve afferents.

In chloralose-anaesthetized cats, sympathetic reflex responses were recorded in left cardiac and renal nerve during stimulation of afferent fibres in the ipsilateral phrenic nerve. In cardiac nerve, a late reflex potential with a mean onset latency of 75.6 +/- 13.8 ms was regularly recorded which, in 20% of the experiments, was preceded by an early, very small reflex component (latency between 35 and 52 ms). In contrast, in renal nerve only a single reflex component after a mean latency of 122.1 +/- 13.1 ms was observed. Bilateral microinjections of the GABA-agonist muscimol into the rostral ventrolateral medulla oblongata resulted in a nearly complete abolition of sympathetic background activity and in an 88% reduction of the late reflex amplitude with only small effects on the latency of the evoked potentials. Under this condition, an early reflex component was never observed to appear. After subsequent high cervical spinalization, the residual small potentials which persisted after bilateral muscimol injections were completely abolished and in cardiac nerve an early reflex potential with a mean latency of 45 +/- 10 ms was observed in all but one experiment. The early reflex was therefore referred to as a spinal reflex component which, however, is suppressed in most animals with an intact neuraxis. In the renal nerve a spinal response was only observed in one experiment after spinalization. The results suggest that sympathetic reflexes evoked by stimulation of phrenic nerve afferent fibres possess similar spinal and supraspinal pathways as previously described for somato-sympathetic and viscero-sympathetic reflexes.(ABSTRACT TRUNCATED AT 250 WORDS)

An intracellular study of the synaptic input to sympathetic preganglionic neurones of the third thoracic segment of the cat.

In chloralose anaesthetized, paralyzed and artificially ventilated cats intracellular recordings were obtained from sympathetic preganglionic neurones (SPN) of the third thoracic segment of the spinal cord identified by antidromic stimulation of the white ramus T3. The synaptic input to SPNs was assessed, in cats with intact neuraxis or spinalized at C3, by electrical stimulation of segmental afferent fibres in intercostal nerves and white rami of adjacent thoracic segments and by stimulation of the ipsi- and contralateral dorsolateral funiculus and of the dorsal root entry zone of the cervical spinal cord. In both preparations SPNs showed on-going synaptic activity which predominantly consisted of excitatory post-synaptic potentials (EPSPs). Inhibitory post-synaptic potentials (IPSPs) were rarely observed. EPSPs were single step (5 mV) or, less frequently, large (up to 20 mV) summation EPSPs. The proportion of SPNs showing very low levels of on-going activity was markedly higher in spinal than in intact cats. Stimulation of somatic and sympathetic afferent fibres evoked early EPSPs (amplitude 3 mV, latency 5-22.3 ms), and late, summation EPSPs (amplitude up to 20 mV, latency 27-55 ms). Early and late EPSPs were evoked in nearly all SPNs in which this synaptic input was tested in the intact preparation (from 79-93% of the SPNs). In spinal cats, early EPSPs were evoked in 88% of the SPNs, whereas late EPSPs were recorded only in half of the neurones. No evidence for a monosynaptic pathway from these segmental afferent fibres to SPNs was obtained. In both intact and spinal cats, stimulation of the dorsolateral funiculus evoked early and late EPSPs in SPNs. Late EPSPs were recorded in 70% and 37% of the SPNs in intact and spinal cats, respectively. Early EPSPs, however, were evoked in all neurones. The early EPSPs evoked by stimulation of the dorsolateral funiculus had several components which are suggested to arise from stimulation of descending excitatory pathways with different conduction velocities. The following conduction velocities were calculated in intact (spinal) cats: 9.5-25 m/s (7.8-13.2 m/s), 5.7-9.5 m/s (5.5-7.8 m/s), 3.8-5.7 m/s (3.2-5.5 m/s), and 2.6-3.8 m/s (2.1-3.2 m/s). EPSPs of these various groups were elicited in a varying percentage in SPNs. EPSPs of the most rapidly conducting pathway were subthreshold for the generation of action potentials; some EPSPs of this group had a constant latency suggesting a monosynaptic pathway to SPNs. Stimulation of the dorsal root entry zone at the cervical level yielded essentially the same results as stimulation of the dorsolateral funiculus.(ABSTRACT TRUNCATED AT 400 WORDS)

Tonic descending inhibition of the spinal cardio-sympathetic reflex in the cat.

Electrical stimulation of the left inferior cardiac nerve elicited a two-component reflex potential (spinal and supraspinal reflexes) in the ipsilateral white ramus T3 from which recordings were made in chloralose-anaesthetised cats. Reversible interruption of all spinal pathways achieved by cooling the spinal cord at C2/C3 produced an enhancement of the spinal reflex and abolished the supraspinal reflex, the latter usually being the more prominent reflex potential prior to spinal cord block. The spinal cord block-induced increase in the amplitude of the spinal reflex was, however, less than the increase observed during stimulation of the somatic intercostal nerve T4. Recordings of the afferent volley following cardiac nerve stimulation and analysis of the stimulus-reflex response relationship in neuraxis-blocked cats indicated that the spinal reflex as determined here was activated by A delta afferent fibres. However, if stimulus strength was raised above C-fibre threshold, spinal cord block revealed in addition a late spinal reflex response. In some cases, the appearance of this late potential was accompanied by a secondary decline of the earlier spinal reflex potential, possibly indicating C-fibre-mediated afferent inhibition. Neither baroreceptor activation nor denervation had any effect on spinal reflex amplitudes. Pharmacologically, clonidine given i.v. to cats with a blocked neuraxis reduced the spinal reflex amplitudes to pre-block values, an action which could be antagonised by the subsequent administration of the alpha 2-adrenoceptor antagonist rauwolscine. When given to non-pretreated cats with intact neuraxis, however, neither rauwolscine nor its analog yohimbine were capable of inducing a persistent release from tonic inhibition. The results suggest that both purely visceral and somato-visceral reflexes are subject to tonic descending inhibition, but they do not support the hypothesis that a catecholamine is the responsible transmitter mediating this inhibition.

Chemoreceptor stimulation on sympathetic activity: dependence on respiratory phase.

Experiments were performed on chloralose-anesthetized, vagotomized, paralyzed, and artificially ventilated cats breathing 100% O2. Peripheral chemoreceptors were stimulated by rapid injections of CO2-saturated NaHCO3 in different phases of the respiratory cycle. Responses of cardiac and renal sympathetic nerves were computed by digital integration. Spontaneous sympathetic activity was consistently modulated by respiration, the modulation being greater for cardiac than for renal nerves. Cardiac nerve responses to peripheral chemoreceptor stimulation depended on the respiratory phase for at least one experimental condition in four of seven animals: the responses were largest during late inspiration and smallest (or absent) during postinspiration and early expiration. Renal nerve responses depended on respiratory phase in only two of eight animals. An average end-tidal CO2 concentration increase from 4.6 +/- 0.8% (SD) to 6.7 +/- 0.9% enhanced the respiratory modulation of spontaneous activity but reduced the responses to peripheral chemoreceptor stimulation. The results indicate that the respiratory modulation of chemoreceptor-induced sympathetic responses was less prominent than the modulation of spontaneous activity. It is hypothesized that the phase dependence of the responses is caused by the spontaneously occurring expiratory diminution of sympathetic activity rather than by an inherent gating of the chemoreceptor reflex.

Central action of alpha-adrenoceptor agents on the baroreceptor reflex.

In chloralose-anaesthetized cats the effects of intravenous application of the alpha 1- and alpha 2-adrenoceptor agonistic and antagonistic agents methoxamine, prazosin, B-HT 933 and rauwolscine were tested on baroreceptor reflex, sympathetic background activity and blood pressure. Sympathetic activity was recorded from the renal nerve and the efficacy of the central transmission of the baroreceptor reflex was measured by the duration of the complete inhibition of renal nerve activity during electrical stimulation of the left carotid sinus nerve. All baroreceptors were denervated by sectioning both carotid sinus and vagal nerves. The alpha 1-agonist methoxamine increased baroreceptor-induced sympatho-inhibition, sympathetic background activity and blood pressure. The alpha 1-antagonist prazosin had the opposite effects. The alpha 2-agonist B-HT 933 was most effective in augmenting the inhibitory response in sympathetic activity to baroreceptor stimulation; sympathetic background activity and blood pressure were also decreased. At low doses (50 micrograms/kg) the alpha 2-antagonist rauwolscine reduced the baroreceptor sympathetic reflex inhibition and increased sympathetic activity and blood pressure. The effect of B-HT 933 upon the baroreceptor reflex could be completely antagonized by rauwolscine. These findings demonstrate a very effective facilitation of the baroreceptor reflex transmission by stimulation of central alpha 2-adrenoceptors. Through such humoral-neuronal interaction circulating catecholamines are likely to modulate cardiovascular control.

K-dependent inhibition in the dentate-CA3 network of guinea pig hippocampal slices.

1. The occurrence of potassium-dependent inhibitory postsynaptic potentials (K-IPSPs) in relation to burst discharges induced by 4-aminopyridine (4-AP; 30 microM) was studied in CA3, granule and hilar neurons in guinea pig hippocampal slices with the use of paired extra- and/or intracellular recording. 2. Slow small (2-5 mV) and large (up to 30 mV) K-IPSPs were observed in CA3, granule and in some hilar neurons during 4-AP applications in the presence of blockers for fast synaptic transmission, picrotoxin (50 microM), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5-10 microM). Amplitudes of K-IPSPs were linearly related to voltage, and they reversed in sign close to -100 mV, as expected for synaptic potentials generated by an increase in K-conductance. 3. In CA3 neurons, 4-AP applied in the presence of picrotoxin elicited burst discharges and K-IPSPs. CNQX blocked the burst discharge activity and increased the amplitude of K-IPSPs. 4. In granule cells, 4-AP applied in the presence of picrotoxin elicited K-IPSPs and only inconsistently small excitatory postsynaptic potentials (EPSPs). The EPSPs were blocked by CNQX, but CNQX application did not affect the K-IPSPs. However, in granule cells it could be observed that blockade of Cl-inhibition by picrotoxin in the presence of CNQX increased the amplitude of K-IPSPs. 5. In hilar neurons, 4-AP applied in the presence of picrotoxin elicited mainly burst discharges. CNQX blocked the burst discharges only in a few cells. In most hilar neurons K-IPSPs were observed at the beginning of the 4-AP effect, but subsequently K-IPSPs were replaced by burst discharges. 6. To determine the type of cells that burst in picrotoxin and 4-AP, neurons were stained intracellularly with horseradish peroxidase. Neurons stained in the granule cell layer did not burst and were morphologically identified as granule cells. Neurons stained in the hilar region burst and were nonpyramidal, nongranule cells. Bursting cells stained in the CA3 area were all pyramidal cells. 7. The hilar neurons varied considerably in size and dendritic organization. They could be classified as aspiny and spiny cells, the latter including mossy cells. 8. We conclude that K-dependent inhibition may explain the long-lasting IPSPs observed in in vivo recordings from hippocampal cells. In a hippocampal lamella, burst discharge activity of hilar neurons including presumed excitatory mossy cells is associated with inhibition of granule cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Tonic descending inhibition of the spinal somato-sympathetic reflex from the lower brain stem.

In chloralose-anaesthetized cats the spinal and supraspinal components of the somato-sympathetic reflex were evoked in the white ramus at T3 and/or L2 by stimulation of intercostal and spinal nerves. A reversible blockade of all ascending and descending spinal pathways was performed by cooling the spinal cord between the second and third cervical segment. Total blockade of conduction was produced at temperatures below 8.5 degrees C (281.5 K). The spinal blockade produced the following reversible effects. (1) Mean arterial pressure fell to 30-50 mm Hg (4.0-6.7 kpa) and the tonic background activity in the white ramus was reduced to 0-24% of control (mean 12.1 +/- 10.0%). (2) The amplitude of the early spinal reflex was increased from 100% to 111-316% (mean 200.9 +/- 49.5%, n = 49) at the thoracic level and to 125-342% (mean 181.4 +/- 74.4%, n = 7) at the lumbar level. The onset latency of the spinal reflex at T3 (range 8-21 msec) was shortened by 0.5-3.0 msec (mean 1.7 +/- 0.9 msec). (3) Supraspinal components were completely abolished. (4) Neither baroreceptor denervation nor midcollicular decerebration altered these effects. (5) The cold block induced increase of the amplitude of the spinal reflex was reduced by the alpha-adrenoceptor agonist clonidine; this effect was reversed by the alpha-adrenoceptor antagonist yohimbine. Selective cooling of the dorsolateral funiculus caused the same effects on the spinal and supraspinal reflexes as cold block of the whole spinal cord. From these findings it is concluded that in the anaesthetized cat the spinal component of the somato-sympathetic reflex is modulated by a descending tonic inhibition. This inhibition acts at both the thoracic and the lumbar level and its origin is in the medulla oblongata. This inhibition is, however, independent of baroreceptor inputs. The pathways descends in the dorsolateral funiculus. It is suggested that noradrenaline or adrenaline might be involved in the transmission of this inhibitory influence.

Neurones within the "chemosensitive area" on the ventral surface of the brainstem which project to the intermediolateral column.

With the method of retrograde transport of horseradish peroxidase it has been demonstrated that neurones within the "chemosensitive area" of the brainstem project to the thoracic intermediolateral column. The function of these neurones is discussed in regard to the regulation of blood pressure.