Intrinsic Pacemaker Properties Research Articles

Electrophysiological Properties of Rostral Ventrolateral Medulla Presympathetic Neurons Modulated by the Respiratory Network in Rats

The respiratory pattern generator modulates the sympathetic outflow, the strength of which is enhanced by challenges produced by hypoxia. This coupling is due to the respiratory-modulated presympathetic neurons in the rostral ventrolateral medulla (RVLM), but the underlining electrophysiological mechanisms remain unclear. For a better understanding of the neural substrates responsible for generation of this respiratory-sympathetic coupling, we combined immunofluorescence, single cell qRT-pCR, and electrophysiological recordings of the RVLM presympathetic neurons in in situ preparations from normal rats and rats submitted to a metabolic challenge produced by chronic intermittent hypoxia (CIH). Our results show that the spinally projected cathecholaminergic C1 and non-C1 respiratory-modulated RVLM presympathetic neurons constitute a heterogeneous neuronal population regarding the intrinsic electrophysiological properties, respiratory synaptic inputs, and expression of ionic currents, albeit all neurons presented persistent sodium current-dependent intrinsic pacemaker properties after synaptic blockade. A specific subpopulation of non-C1 respiratory-modulated RVLM presympathetic neurons presented enhanced excitatory synaptic inputs from the respiratory network after CIH. This phenomenon may contribute to the increased sympathetic activity observed in CIH rats. We conclude that the different respiratory-modulated RVLM presympathetic neurons contribute to the central generation of respiratory-sympathetic coupling as part of a complex neuronal network, which in response to the challenges produced by CIH contribute to respiratory-related increase in the sympathetic activity.

Determinants of inspiratory activity

In vitro and in vivo studies have identified the pre-Bötzinger complex as an important kernel for the generation of inspiratory activity. The mechanisms underlying inspiratory rhythm generation involve pacemaker as well as synaptic mechanisms. In slice preparations, blockade of pacemaker properties with blockers for the persistent Na+ current, and the Ca2+-activated inward cationic current, abolishes respiratory activity. Here we show that blockade of the persistent Na+ current alone is sufficient to abolish respiratory activity in the in situ preparation. Although pacemaker neurons may be critical for establishing the basic respiratory rhythm, their rhythmic output is modulated by many elements of the respiratory network. For example, levels of synaptic inhibition control whether they burst or not, and endogenously released neuromodulators, such as serotonin and substance P modulate their intrinsic membrane currents. We hypothesize that the balance between synaptic and intrinsic pacemaker properties in the respiratory network is plastic, and that alterations of this balance may lead to dynamic reconfigurations of the respiratory network, which ultimately give rise to different activity patterns.

"Rapid" rhythmic discharges of sympathetic nerves: sources, mechanisms of generation, and physiological relevance.

Like virtually all other physiological control systems, the sympathetic nervous system controlling cardiovascular function is characterized by the presence of rhythmic activity. These include slow rhythms with frequencies at or below that of the respiration and rapid rhythms with frequencies at or above that of the heart beat. The rapid rhythms are the subject of this review. The specific questions entertained are as follows: (1) Are the rapid cardiac-related and 10-Hz rhythms inherent to central sympathetic networks, or are they imposed on sympathetic nerve discharge (SND) by extrinsic periodic inputs? (2) Does basal SND arise from an anatomically circumscribed "vasomotor center" composed of pacemaker neurons in the rostral ventrolateral medulla or from an anatomically distributed network oscillator composed of different types of brainstem neurons, none of which necessarily have intrinsic pacemaker properties? (3) Are the rapid rhythms generated by single circuits or by systems of coupled oscillators, each with a separate target? (4) Are the rapid rhythms in SND simply by-products of the sympathetic generating mechanisms, or do they subserve selective and special functions, such as the formulation of differential patterns of spinal sympathetic outflow that support particular behaviors? The controversial aspects of these issues and the state-of-the-art analytical methods used to study them are stressed in this review.

Scheduled activity reorganizes circadian phase of Syrian hamsters under full and skeleton photoperiods

Circadian rhythms can be shifted or entrained by light and by arousing nonphotic stimuli. Interactions between photic and nonphotic stimuli were examined by subjecting hamsters to a daily 3 h bout of induced running under full (FPP) or skeleton (two daily light pulses; SPP) photoperiods. Activity scheduled in mid-day of a FPP induced large phase delays (260±63 min) in hamsters that ran more than 4000 rev/3 h. Split rhythms were not evident in constant dark (DD) tests. Activity scheduled in mid-subjective day of a SPP induced 180° inversions of circadian phase, apparently achieved by oscillator splitting in some cases. Activity scheduled late-day and early-night induced a mix of phase delays, advances and no responses. Activity scheduled at two phase ranges late in the night had no effect, but scheduled 1 h later (beginning the last hour of darkness) induced large phase delays (238±30 min). There was no evidence of oscillator splitting during DD tests, but free-running period was significantly longer in groups that showed large phase delays. Induced running schedules have powerful effects on the phase of photic entrainment and can alter intrinsic pacemaker properties, including internal oscillator coupling and period.

Properties of presympathetic neurones in the rostral ventrolateral medulla in the rat: an intracellular study "in vivo'.

1. Intracellular recordings were made in pentobarbitone-anaesthetized rats from sixty-eight neurones located in the rostral ventrolateral medulla (RVLM), which responded with inhibition (latency, 33.6 +/- 9.3 ms) after stimulation of the aortic depressor nerve with short bursts of pulses. This inhibition was due to chloride- and voltage-dependent IPSPs. 2. Seventeen neurones could be excited antidromically after stimulation in the T2 spinal segment (conduction velocity 1.9-8.5 m.s-1) and were classified as RVLM presympathetic vasomotor neurones. 3. "Spontaneously' active neurones (n = 29) displayed a largely irregular pattern of firing, with no clear relationship between the level of the membrane potential and cycles of phrenic nerve activity at end-tidal CO2 < 5.0%. Cardiac cycle-related shifts of the membrane potential were not considered indicative of baroreceptor input as they could be due to movement artifacts. 4. All neurones displayed large synaptic activity (EPSPs and IPSPs, peak-to-peak amplitude > 5.0 mV). The depolarizing IPSPs observed during injection of chloride and/or negative current consisted of a phasic and a tonic component. 5. The on-going activity of these neurones resulted from synaptic inputs, with individual action potentials usually preceded by identifiable fast EPSPs. 6. No evidence was found for the presence of gradual depolarizations (autodepolarizations) between individual action potentials, and therefore under these experimental conditions the activity of RVLM presympathetic neurones did not depend on intrinsic pacemaker properties. 7. These results are consistent with the "network' hypothesis for the generation of sympathetic vasomotor tone.

C1 neurons of neonatal rats: intrinsic beating properties and alpha 2-adrenergic receptors

Tyrosine-hydroxylase-immunoreactive (TH-ir) neurons (C1 cells) of the rostral ventrolateral medulla (RVL) control sympathetic tone and may be inhibited by the hypotensive drug clonidine. The present study seeks to describe some of the intrinsic properties of bulbospinal C1 cells and to establish whether they have alpha 2-adrenergic inhibitory "autoreceptors." RVL bulbospinal cells (retrogradely labeled with microbeads) were recorded in thin slices from 3- to 7-day-old rats with patch electrodes containing Lucifer yellow. Forty-two of eighty-nine cells were recovered histologically, and 69% were TH-ir. The properties of TH-ir cells could not be distinguished from those of the other bulbospinal cells. Most cells were spontaneously active (0.5-5 spikes/s). Blockade of fast postsynaptic potentials with kynurenic acid plus bicuculline plus strychnine or with a low-Ca(2+)-high-Mg2+ medium failed to reduce their firing rate. The membrane trajectory consisted of one of two patterns interconvertible by injections of depolarizing or hyperpolarizing current: 1) depolarizing ramps leading to spikes or 2) slow oscillations (tetrodotoxin resistant but absent at hyperpolarized potentials) leading to spikes or repolarization. The cells had Ca(2+)-dependent afterhyperpolarizations, large transient outward currents, and a hyperpolarization-activated inward current activated at potentials less than -65 mV, blocked by CsCl, and presumably responsible for a large inward rectification. Of 27 TH-ir cells, 21 had inhibitory responses to alpha 2-adrenergic agonists. These were blocked by idazoxan (n = 9). alpha 2-Agonists activated an inwardly rectifying potassium conductance (KIR). We confirm that bulbospinal C1 cells have intrinsic pacemaker properties and demonstrate that they have alpha 2-adrenergic receptors (autoreceptors) coupled to KIR channels.

Morphology of rostral medullary neurons with intrinsic pacemaker activity in the rat

Neurons with regular ongoing activity attributable to intrinsic pacemaker properties were recorded in coronal tissue slices within the nucleus reticularis rostroventrolateralis of the rat medulla oblongata (RVL). The cells were injected with horseradish peroxidase or Lucifer yellow and their dendritic and proximal axonal characteristics were investigated ( n=15). These small-to-medium-sized neurons had a simple dendritic arborization (3–6 primary dendrites branching up to 3 times) apparently confined within the limits of nucleus RVL and with limited extension in the rostrocaudal direction. Their axons originated either from the cell body or from a primary dendrite and coursed in a dorsomedial direction without giving rise to lical arborizations. It is concluded that RVL pacemaker neurons, presumed to represent a non-adrenergic class of sympathoexcitatory premotor neurons, exhibit characteristics reminiscent of the archetypal ‘reticular core’ neurons.

Slow excitatory amino acid receptor-mediated synaptic transmission in turtle cerebellar Purkinje cells

1. The excitatory synaptic responses of turtle Purkinje cells to climbing and parallel fiber (CF and PF) stimulation have been studied by the use of intrasomatic and intradendritic recordings in intact cerebellum and brain stem-cerebellum preparations in vitro. 2. Activation of CF inputs from the cerebellar peduncle or the region of the inferior olive evoked complex spikes followed by slow excitatory postsynaptic potentials (EPSPs), both of which were evoked in an all-or-none fashion. 3. Single stimuli applied to the cerebellar molecular layer activated fast PF-mediated EPSPs; brief trains of PF stimuli (2-5 stimuli, 50-100 Hz) evoked volleys of fast EPSPs followed by a slow, long-lasting EPSP. The amplitude of the fast and slow PF-mediated EPSPs were both graded with stimulus intensity. 4. Slow EPSPs evoked both by CF and PF stimulation were associated with an increase in membrane conductance and were increased in amplitude by hyperpolarization. 5. The CF-evoked slow EPSP was profoundly attenuated by repetitive activation at interstimulus intervals of less than 15-20 s, whereas the PF-evoked slow EPSP was not reduced by repetitive activation. 6. The PF-evoked slow EPSP readily triggered dendritic pacemaker discharges when activated at or near resting membrane potential. The activation of this potential by phasic PF volleys may, therefore, provide an appropriate synaptic drive to cerebellar Purkinje cells to entrain the intrinsic pacemaker properties of these cells to cycles of motor activity. 7. Both slow synaptic potentials were blocked by the excitatory amino acid antagonists kynurenate and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not by DL-2-amino-5-phosphonovalerate (DL-AP5) or L-serine-O-phosphate (L-SOP). The PF-evoked slow EPSP was selectively antagonized by L-2-amino-4-phosphonobutyrate (L-AP4; 20-100 microM). 8. It is suggested that the CF- and PF-evoked slow EPSPs observed in this study represent a novel class of excitatory amino acid receptor-mediated slow synaptic potentials activated by Purkinje cell afferents, which may play a role in synaptic integration and motor pattern generation in the cerebellum.

Rostral ventrolateral medullary neurons with intrinsic pacemaker properties are not catecholaminergic

Neurons of the rat rostral ventrolateral medulla with intrinsic pacemaker properties and immediately adjacent silent neurons were recorded in tissue slices, labeled intracellularly with the fluorescent dye Lucifer yellow, and the tissue was subsequently processed for the immunocytochemical detection of the catecholamine-synthesizing enzyme tyrosine-hydroxylase. Forty-five percent (9/20) of the silent neurons were tyrosine hydroxylase-immunoreactive, but none of the pacemaker cells were (0/15). We conclude that the ventrolateral reticular neurons with intrinsic pacemaker properties are not the C 1 adrenergic cells.

Reticulospinal pacemaker neurons of the rat rostral ventrolateral medulla with putative sympathoexcitatory function: an intracellular study in vitro

Extra- and intracellular recordings of tonically active neurons were obtained in slices of the rat rostral ventrolateral medulla maintained at 31°C. The predominant type consisted of cells with a regular non-bursting discharge rate of 9 ± 0.3 spikes/s (mean ± S.E.M., n = 84). Intracellular recordings revealed that these neurons ( n = 43) exhibited typical pacemaker potentials reset after a single spike, and an input resistance of 138 ± 10 MΩ (n = 21) . No excitatory postsynaptic potentials were detected even during hyperpolarization (5–10 mV) which invariably resulted in silencing the cells ( n = 28). Eighteen cells were injected intracellularly with Lucifer yellow, and the tissue was subsequently processed for the immunohistochemical detection of the adrenergic marker phenylethanolamine N-methyltransferase (PNMT). None of the 12 dye-marked cells recovered exhibited any PNMT-like immunoreactivity, but all were surrounded by numerous adrenergic neurons. In 7 rats subjected to intraspinal injections (T 3) of rhodamine-tagged microbeads. 4 out of 9 pacemaker cells marked intracellularly with Lucifer yellow were found labeled with the retrograde marker. It is concluded that the rostral ventrolateral medulla contains non-adrenergic reticulospinal cells with intrinsic pacemaker properties. These neurons probably represent a group of sympathoexcitatory cells on which the basal sympathetic tone depends.

Impulse propagation from the SA-node to the ventricles.

Normally the pacemaker of the mammalian heart is located in the sinus node. In the rabbit the sinus node can be subdivided into two regions, the center of the node where the impulse originates and the border zone through which the impulse is conducted towards the atrium. Conduction properties of both regions were investigated. It appeared that conduction velocity increases and refractoriness decreases when one goes from the nodal center towards the atrium. The tissue mass of the atrium is large in comparison to the sinus node and normally the resting membrane potential of atrial fibers is more negative than that of nodal fibers; consequently, a potential difference exists causing a current flow between both areas. Evidently this hyperpolarizing current flow depresses impulse formation in the border zone fibers which have better intrinsic pacemaker properties than fibers in the nodal center. If the impulse has reached the atrium it is conducted with a relatively high safety factor and will reach the AV node in principle without difficulty. The AV node, if deprived of sinus nodal dominance, develops spontaneous activity originating from the lower nodal fibers. Also in this structure, electrotonic depression by surrounding tissue causes deceleration of the pacemaker.

The influence of the atrial myocardium on impulse formation in the rabbit sinus node.

In the isolated right atrium of the rabbit heart the influence of the atrial myocardium on impulse formation in the sinus node was investigated. Under normal conditions the pacemaker (earliest activation) was located in the center of the node where fibers with the highest rate of diastolic depolarization were found. After disconnection of the atrium from the sinus node spontaneous cycle length decreased from a mean of 348 ms to a mean of 288 ms (-18%) in all experiments (n = 15). This was accompanied by a shift of the pacemaker from the nodal center towards the border zone. By means of multiple microelectrode impalements changes in action potential configuration were studied. After disconnection of atrium and sinus node the rate of diastolic depolarization of fibers in the border zone was increased from a mean of 26 mV/s to a mean of 78 mV/s, whereas in the center of the sinus node no increase was found (mean: 52 mV/s). It was concluded that the fibers in the border zone of the sinus node are better pacemaker fibers than in the nodal center. However under normal conditions the intrinsic pacemaker properties of the border zone fibers are electronically depressed by the connected atrial myocardium.

Autonomic blockade and sick sinus syndrome. New concept in the interpretation of electrophysiological and Holter data.

Autonomic blockade is commonly employed as a test of sinus node dysfunction. We compared primary and secondary atrial postpacing pauses and postextrasystolic sino-atrial responses before and after autonomic blockade in 56 patients with the clinical diagnosis of sick sinus syndrome. Pharmacological autonomic blockade was achieved by atropine 0.04 mg kg-1 and propranolol 0.2 mg kg-1, i.v. In a group of patients with a normal intrinsic heart rate the number of positive electrophysiologic variables associated with sinus node dysfunction declined after autonomic blockade. In 91% of these patients, sinus node function was characterized by a normal intrinsic recovery time, gradual exponential return to the constant sinus cycle length, and biphasic postextrasystolic return responses. Three patients in this group had intrinsic SA-block revealed by atrial pacing and verified by Holter monitoring. Besides normal intrinsic pacemaker properties in 53% of patients, rhythm monitoring revealed severe sinus node dysfunction as manifested by bradycardia and the tachycardia-bradycardia syndrome. SA-block and sinus arrest up to 29120 ms. In the abnormal intrinsic heart rate group, disturbed intrinsic rhythmicity was characterized in all by a prolonged corrected intrinsic recovery time (2320 +/- 2740 ms [+/- SD]), arrhythmia and/or bradycardia in the secondary postpacing cycles, chaotic postextrasystolic patterns, or prolonged sinoatrial conduction times. Significantly slow minimal heart rates during sleep significantly prolonged average sinus cycle lengths and positive ECGs for sinoatrial disorders in the waking period were present on the 24-h rhythm recording. It is concluded that intrinsic heart rate obtained by autonomic blockade is the best and most simple method for the diagnosis of intrinsic sinus node dysfunction. Combined autonomic blockade and electrophysiological tests can be of great value in unmasking the severity and degree of intrinsic dysfunction and analyzing the abnormality of secondary pacemaker function. These investigations, however, are rather insensitive and therefore ineffective in detecting autonomic sinus node dysfunction. To assess the role and significance of the autonomous neurovegetative tone in the genesis of sinoatrial disorders, rhythm monitoring is required.