Stochastic behavior of atrial and ventricular intrinsic cardiac neurons.

To quantify the concurrent transduction capabilities of spatially distributed intrinsic cardiac neurons, the activities generated by atrial vs. ventricular intrinsic cardiac neurons were recorded simultaneously in 12 anesthetized dogs at baseline and during alterations in the cardiac milieu. Few (3%) identified atrial and ventricular neurons (2 of 72 characterized neurons) responded solely to regional mechanical deformation, doing so in a tightly coupled fashion (cross-correlation coefficient r = 0.63). The remaining (97%) atrial and ventricular neurons transduced multimodal stimuli to display stochastic behavior. Specifically, ventricular chemosensory inputs modified these populations such that they generated no short-term coherence among their activities (cross-correlation coefficient r = 0.21 +/- 0.07). Regional ventricular ischemia activated most atrial and ventricular neurons in a noncoupled fashion. Nicotinic activation of atrial neurons enhanced ventricular neuronal activity. Acute decentralization of the intrinsic cardiac nervous system obtunded its neuron responsiveness to cardiac sensory stimuli. Most atrial and ventricular intrinsic cardiac neurons generate concurrent stochastic activity that is predicated primarily upon their cardiac chemotransduction. As a consequence, they display relative independent short-term (beat-to-beat) control over regional cardiac indexes. Over longer time scales, their functional interdependence is manifest as the result of interganglionic interconnections and descending inputs.     

Efferent neurons of the lateral-line system and the VIII cranial nerve in the brainstem of anurans

Summary The octavo-lateral efferent system of several anuran species was studied by means of retrograde transport of horseradish peroxidase. This system is organized similarly in all larval anurans and in all adult aglossids. All have two groups of efferent neurons in the nucleus reticularis medialis between the VIIIth and the IXth motor nucleus. The caudal group consists of efferent neurons that supply the posterior lateral-line nerve (NLLp) and a considerably smaller group of neurons supplying both the NLLp and the anterior lateral-line nerve (NLLa). The rostral group is composed of efferent neurons supplying the NLLa, neurons projecting to the inner ear and neurons supplying both the inner ear and the NLLa. Efferent neurons of the VIIIth cranial nerve exhibit a rostrocaudal cytoarchitectonic differentiation. Caudal perikarya, which are rounder in shape than those of the rostral part, have a dendritic projection to the superior olive. It is suggested that this differentiation reflects a functional differentiation of acoustic and vestibular efferent neurons.Labeled neurons were ipsilateral to the site of application of HRP. None were found in the vestibular nuclei or in the cerebellum.Efferent axons projecting to neuromasts of the NLLa leave the medulla with the VIIth nerve, axons projecting to neuromasts of the NLLp exit via the IXth nerve. Cell counts and the observation of axonal branching revealed that efferent units of both the lateral-line and the VIIIth-nerve system supply more than one receptor organ. In contrast to the lateral-line system, dendrites of efferent neurons of the VIIIth nerve project dorsally onto its nuclei, and afferents of the VIIIth nerve project onto efferent neurons. These structures most probably represent a feedback loop between the afferent and efferent systems of the VIIIth cranial nerve.     

Intrinsic cardiac nervous system in tachycardia induced heart failure

The purpose of this study was to test the hypothesis that early-stage heart failure differentially affects the intrinsic cardiac nervous system's capacity to regulate cardiac function. After 2 wk of rapid ventricular pacing in nine anesthetized canines, cardiac and right atrial neuronal function were evaluated in situ in response to enhanced cardiac sensory inputs, stimulation of extracardiac autonomic efferent neuronal inputs, and close coronary arterial administration of neurochemicals that included nicotine. Right atrial neuronal intracellular electrophysiological properties were then evaluated in vitro in response to synaptic activation and nicotine. Intrinsic cardiac nicotine-sensitive, neuronally induced cardiac responses were also evaluated in eight sham-operated, unpaced animals. Two weeks of rapid ventricular pacing reduced the cardiac index by 54%. Intrinsic cardiac neurons of paced hearts maintained their cardiac mechano- and chemosensory transduction properties in vivo. They also responded normally to sympathetic and parasympathetic preganglionic efferent neuronal inputs, as well as to locally administered alpha-or beta-adrenergic agonists or angiotensin II. The dose of nicotine needed to modify intrinsic cardiac neurons was 50 times greater in failure compared with normal preparations. That dose failed to alter monitored cardiovascular indexes in failing preparations. Phasic and accommodating neurons identified in vitro displayed altered intracellular membrane properties compared with control, including decreased membrane resistance, indicative of reduced excitability. Early-stage heart failure differentially affects the intrinsic cardiac nervous system's capacity to regulate cardiodynamics. While maintaining its capacity to transduce cardiac mechano- and chemosensory inputs, as well as inputs from extracardiac autonomic efferent neurons, intrinsic cardiac nicotine-sensitive, local-circuit neurons differentially remodel such that their capacity to influence cardiodynamics becomes obtunded.     

A Simple and Reliable Technique to Combine Oligonucleotide Probe in Situ Hybridization with Neuronal Tract Tracing in Vertebrate Embryos

We describe a simple and reliable combination of in situ hybridization with neuronal tracing. The technique uses recent advances in the field of neuronal tract tracing including fast diffusing, low molecular weight dextran amines and fade resistant fluorescent dyes, and combines them with in situ hybridization using a sensitive Oligonucleotide probe. Using this technique we have investigated the mRNA encoding the trkB receptor for brain-derived neurotrophic factor in identified facial and vestibular afferent and efferent neurons. We found very low levels of trkB mRNA in facial efferent neurons, whereas in the vestibular afferent neurons, clear labeling for the trkB mRNA could be seen. This technique can be applied to the developing embryo to study topology of a variety of cellular markers with reference to neuronal population or fibers identified by their origin or target.     

Neural organization of the circadian system of the cockroach Leucophaea maderae

The cockroach Leucophaea maderae was the first animal in which lesion experiments localized an endogenous circadian clock to a particular brain area, the optic lobe. The neural organization of the circadian system, however, including entrainment pathways, coupling elements of the bilaterally distributed internal clock, and output pathways controlling circadian locomotor rhythms are only recently beginning to be elucidated. As in flies and other insect species, pigment-dispersing hormone (PDH)-immunoreac- tive neurons of the accessory medulla of the cockroach are crucial elements of the circadian system. Lesions and transplantation experiments showed that the endogeneous circadian clock of the brain resides in neurons associated with the accessory medulla. The accessory medulla is organized into a nodular core receiving photic input, and into internodular and peripheral neuropil involved in efferent output and coupling input. Photic entrainment of the clock through compound eye photoreceptors appears to occur via parallel, indirect pathways through the medulla. Light-like phase shifts in circadian locomotor activity after injections of γ-aminobutyric acid (GABA)- or Mas-allatotropin into the vicinity of the accessory medulla suggest that both substances are involved in photic entrainment. Extraocular, cryptochrome-based photoreceptors appear to be present in the optic lobe, but their role in photic entrainment has not been examined. Pigment-dispersing hormone-immunoreactive neurons provide efferent output from the accessory medulla to several brain areas and to the peripheral visual system. Pigment-dispersing hormone-immunoreactive neurons, and additional heterolateral neurons are, furthermore, involved in bilateral coupling of the two pacemakers. The neuronal organization, as well as the prominent involvement of GABA and neuropeptides, shows striking similarities to the organization of the suprachiasmatic nucleus, the circadian clock of the mammalian brain.     

Activity of in situ middle cervical ganglion neurons in dogs, using extracellular recording techniques

Neuronal activity in the in situ middle cervical ganglion of dogs was investigated using extracellular recording techniques. The recorded action potentials were frequently active during specific phases of the cardiac cycle, particularly during systole, and this activity persisted following acute decentralization of the ganglion. The activity of these action potentials was modified when systemic arterial pressure was altered by isoproterenol, noradrenaline, adrenaline, or partial occlusion of the aorta, whether in the intact or acutely decentralized preparation. These neurons were active between systolic pressures of 70 and 180 mmHg (1 mmHg = 133.322 Pa). Action potentials were frequently modified by mechanical distortion of the superior vena cava, ventricular epicardium, or adventitia of the aorta, whether the preparation was acutely decentralized or not. Seventy percent of these action potentials were unaffected by stimulation (1 ms, 4 V, 0.5 Hz) of a cardiopulmonary nerve and 27% were suppressed by such stimulation. Five of the neurons were activated by such stimulation. It is presumed that the latter neurons had axons in a cardiopulmonary nerve and most likely were efferent sympathetic postganglionic neurons. Sixty-three percent of these spontaneously active phase-locked units were modified by stimulation of a ramus or an ansa. It is postulated that some of the neurons in the middle cervical ganglia can be modified by afferent axons arising from receptors in thoracic organs, in particular from the great vessels and heart, whether in an intact or acutely decentralized preparation. The majority of these neurons are presumed not to be afferent neurons or efferent postganglionic neurons, as they are not activated directly by electrical stimulation of axons in cardiopulmonary nerves. Rather they are presumed to be interneurons. These results lend support to the thesis that considerable integration of neuronal activity related to thoracic cardiovascular dynamics occurs within the middle cervical ganglia of dogs.     

Principle of neuronal organization of defensive reflexes in mollusks

Spontaneous and evoked synaptic activity of command neurons for the defensive response of spiracle closing were studied by simultaneous intracellular recording of activity of several identified CNS neurons in snails. Comparison of monosynaptic EPSPs in command neurons evoked by discharges of presynaptic neurons with spontaneous synaptic potentials indicated that the central organization of the defensive reflex is in the form of a two-layered neuron net in which each neuron of the afferent layer possesses a local receptive field, but which overlaps with other afferent neurons. Each neuron of the afferent layer is connected with each neuron of the efferent layer by monosynaptic excitatory connections that differ in efficiency (maximal only with one neuron of the efferent layer). Both receptive fields of neurons of the afferent layer and "fields of efficiency of synaptic connections" are distributed according to the normal law. As a result of this organization the neuron net acquires a new quality: The action of different stimuli leads to the appearance of differently located "spatial excitation profiles" of efferent layer neurons even when this action of the stimulus occurs not at the center of the receptive field.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 16, No. 1, pp. 26–34, January-February, 1984.     

The role of sensory neurons in cervical ripening: effects of estrogen and neuropeptides.

Central nervous system nuclei and circuits, such as the medial preoptic, ventromedial and paraventricular nuclei of the hypothalamus, play important roles in reproduction and parturition, and are influenced by estrogen. Peripheral autonomic and sensory neurons also play important roles in pregnancy and parturition. Moreover, the steroid hormone estrogen acts directly, not only on the reproductive tract organs (uterus and cervix), but also on the central and peripheral nerves by regulating expression of various neuronal genes. The peripheral primary afferent neurons innervating the uterine cervix relay mechanical and biochemical sensory information induced by local cervical events and by passage of fetuses, to the spinal cord and supraspinal centers. Consequently, the birth process in mammals is influenced by the combined action of neurons and hormones. Peripheral sensory stimuli, induced physiologically by fetal expulsion or mechanically by vaginocervical stimulation, alter behavior, as well as autonomic and neuroendocrine systems. Recent evidence indicates that primary afferent neurons innervating the cervix, in addition to their sensory effects, likely exert local "efferent" actions on the ripening cervix near term. These efferent effects may involve estrogen-regulated production of such neuropeptides as substance P and calcitonin gene-related peptide in lumbosacral dorsal root ganglia, and their release in the cervix. Collectively, these findings suggest an interrelationship among estrogen, cervix-related sensory neurons, and local cervical events near term.     

Thermoregulation: some concepts have changed. Functional architecture of the thermoregulatory system

While summarizing the current understanding of how body temperature (T(b)) is regulated, this review discusses the recent progress in the following areas: central and peripheral thermosensitivity and temperature-activated transient receptor potential (TRP) channels; afferent neuronal pathways from peripheral thermosensors; and efferent thermoeffector pathways. It is proposed that activation of temperature-sensitive TRP channels is a mechanism of peripheral thermosensitivity. Special attention is paid to the functional architecture of the thermoregulatory system. The notion that deep T(b) is regulated by a unified system with a single controller is rejected. It is proposed that T(b) is regulated by independent thermoeffector loops, each having its own afferent and efferent branches. The activity of each thermoeffector is triggered by a unique combination of shell and core T(b)s. Temperature-dependent phase transitions in thermosensory neurons cause sequential activation of all neurons of the corresponding thermoeffector loop and eventually a thermoeffector response. No computation of an integrated T(b) or its comparison with an obvious or hidden set point of a unified system is necessary. Coordination between thermoeffectors is achieved through their common controlled variable, T(b). The described model incorporates Kobayashi's views, but Kobayashi's proposal to eliminate the term sensor is rejected. A case against the term set point is also made. Because this term is historically associated with a unified control system, it is more misleading than informative. The term balance point is proposed to designate the regulated level of T(b) and to attract attention to the multiple feedback, feedforward, and open-loop components that contribute to thermal balance.     

Neuronal organization of the avian paraventricular nucleus: intrinsic, afferent, and efferent connections

The heterogeneous paraventricular nucleus (PVN) of birds offers favorable conditions for the analysis of intrinsic, afferent, and efferent connections of neuroendocrine systems. Paraventricular neurons are successfully impregnated with the Golgi-technique. The findings indicate a direct influence of the cerebrospinal fluid (CSF) on the magnocellular neurons that, via their axon terminals in the neural lobe of the pituitary, are also exposed to the hemal milieu. The magnocellular neurons are intermingled with parvocellular elements which may represent local interneurons. A group of parvocellular nerve cells is identified as CSF-contacting neurons. This type of cell forms a basic morphologic component of the avian neuroendocrine apparatus. Immunocytochemical and ultrastructural studies further support the concept of neuronal interactions between parvocellular and magnocellular elements. Moreover, these findings speak in favor of the existence of recurrent collaterals of the magnocellular neurons. Nerve cells giving rise to afferent connections to the PVN are located in the limbic system and autonomic areas of the upper and lower brainstem. Further afferents may originate from the subfornical organ, the organon vasculosum laminae terminalis, the ventral tegmentum, and the area postrema. Via efferent projections, the PVN is connected to the nucleus accumbens, lateral septum, several hypothalamic nuclei, the neural lobe of the pituitary, the organon vasculosum laminae terminalis, the subfornical organ, the pineal organ, the area postrema, the lateral habenular complex, and various autonomic areas of the reticular formation in the upper and lower brainstem and the spinal cord. In conclusion, the PVN may be regarded as an integral component of the neuroendocrine apparatus reciprocally coupled to the limbic system, several circumventricular organs, and various autonomic centers of the brain.     

Light-addressed single-neuron stimulation in dissociated neuronal cultures with sparse expression of ChR2

Individual neurons are heterogeneous and have profound impact on population activity in a complex cortical network. Precise experimental control of the firing of multiple neurons would be therefore beneficial to advance our understanding of cell-network interactions. Except for direct intracellular stimulation, however, it is difficult to gain precise control of targeted neurons without inducing antidromic activation of untargeted neurons. To overcome this problem, we attempt to create a sparse group of photosensitized neurons via transfection of Channelrhodopsin-2 (ChR2) in primary dissociated cultures and then deliver light-addressed stimulation exclusively to these target neurons. We first show that liposome transfection was able to express ChR2 in 0.3-1.9% of cells plated depending on cell density. This spatially sparse but robust expression in our neuronal cultures offered the capability of single cell activation by illuminating a spot of light. We then demonstrated that delivering a pulsed train to photo-activate a single neuron had a substantial effect on the activity level of an entire neuronal culture. Furthermore, the activity level was controllable by altering the frequency of light illumination when 4 neurons were recruited as stimulation targets. These results suggest that organized activation of a very small population of neurons can provide better control over global activity of neuronal circuits than can single-neuron activities by themselves.     

c-Fos expression in rat brain stem and spinal cord in response to activation of cardiac ischemia-sensitive afferent neurons and electrostimulatory modulation

The purpose of this study was to identify central neuronal sites activated by stimulation of cardiac ischemia-sensitive afferent neurons and determine whether electrical stimulation of left vagal afferent fibers modified the pattern of neuronal activation. Fos-like immunoreactivity (Fos-LI) was used as an index of neuronal activation in selected levels of cervical and thoracic spinal cord and brain stem. Adult Sprague-Dawley rats were anesthetized with urethane and underwent intrapericardial infusion of an "inflammatory exudate solution" (IES) containing algogenic substances that are released during ischemia (10 mM adenosine, bradykinin, prostaglandin E2, and 5-hydroxytryptamine) or occlusion of the left anterior descending coronary artery (CoAO) to activate cardiac ischemia-sensitive (nociceptive) afferent fibers. IES and CoAO increased Fos-LI above resting levels in dorsal horns in laminae I-V at C2 and T4 and in the caudal nucleus tractus solitarius. Dorsal rhizotomy virtually eliminated Fos-LI in the spinal cord as well as the brain stem. Neuromodulation of the ischemic signal by electrical stimulation of the central end of the left thoracic vagus excited neurons at the cervical and brain stem level but inhibited neurons at the thoracic spinal cord during IES or CoAO. These results suggest that stimulation of the left thoracic vagus excites descending inhibitory pathways. Inhibition at the thoracic spinal level that suppresses the ischemic (nociceptive) input signal may occur by a short-loop descending pathway via signals from cervical propriospinal circuits and/or a longer-loop descending pathway via signals from the nucleus tractus solitarius.     

Myocardial ischemia induces the release of substance P from cardiac afferent neurons in rat thoracic spinal cord

Antibody-coated microprobes were inserted into the thoracic (T3-4) spinal cord in urethane-anesthetized Sprague-Dawley rats to detect the differences in the release of immunoreactive substance P-like (irSP) substances in response to differential activation of cardiac nociceptive sensory neurons (CNAN). CNAN were stimulated either by intrapericardial infusion of an inflammatory ischemic exudate solution (IES) containing algogenic substances (i.e., 10 mM each of adenosine, bradykinin, prostaglandin E2, and 5-hydroxytryptamine), or by transient occlusion of the left anterior descending coronary artery (CoAO). There was widespread basal release of irSP from the thoracic spinal cord. Stimulation of the CNAN by IES did not alter the pattern of release of irSP. Conversely, CoAO augmented the release of irSP from T3-4 spinal segments from laminae I-VII. This CoAO-induced irSP release was eliminated after thoracic dorsal rhizotomy. These results indicate that heterogeneous activation of cardiac afferents, as with focal coronary artery occlusion, represents an optimum input for activation of the cardiac neuronal hierarchy and for the resultant perception of angina. Excessive stimulation of cardiac nociceptive afferent neurons elicited during regional coronary artery occlusion involves the release of SP in the thoracic spinal cord and suggests that local spinal cord release of SP may be involved in the neural signaling of angina.     

DISTRIBUTION OF ACETYLCHOLINE, CHOLINE, CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN REGIONS AND SINGLE IDENTIFIED AXONS OF THE LOBSTER NERVOUS SYSTEM

Abstract— Acetylcholine, its precursor (choline), and the enzymes of its biosynthesis and degradation (choline acetyltransferase and acetylcholinesterase, respectively) have been studied and quantified in extracts of several regions of the nervous system of the lobster and in single, isolated axons of identified efferent excitatory, efferent inhibitory and afferent sensory neurons. The choline acetyltransferase is a soluble enzyme similar to that from other species. The predominant acetylcholine-hydrolysing enzyme is largely membrane-bound and has been characterized as a specific acetylcholinesterase. A single peak of acetylcholinesterase activity can be detected upon velocity sedimentation analysis of Triton X-100-treated extracts of all regions of the nervous system. Choline acetyltransferase distribution parallels that of sensory neural elements, and its specific activity shows nearly a 500-fold difference from the richest to the poorest neural source. Acetylcholinesterase levels span only a 23-fold range, and activity is found in all neural regions, including those free of known sensory components. A radiochemical microassay for choline and acetylcholine in the range of 20–2000 pmol is described in detail. All 3 types of axons contain comparable levels of choline ( ca. 2 pmol/μg protein), but acetylcholine is asymmetrically distributed. Efferent axons contain no detectable acetylcholine, while sensory axons from abdominal muscle receptor organs have an average of 1·9 pmol/μg protein. Choline acetyltransferase is similarly distributed; sensory axons show at least 500-fold greater activity than efferent axons. Acetylcholinesterase is nearly uniformly distributed among the three types of fibres. These results are discussed in terms of a general view of transmitter accumulation in single neurons.     

Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons

Behavior is a manifestation of temporally and spatially defined neuronal activities. To understand how behavior is controlled by the nervous system, it is important to identify the neuronal substrates responsible for these activities, and to elucidate how they are integrated into a functional circuit. I introduce a novel and general method to conditionally perturb anatomically defined neurons in intact Drosophila. In this method, a temperature-sensitive allele of shibire (shi(ts1)) is overexpressed in neuronal subsets using the GAL4/UAS system. Because the shi gene product is essential for synaptic vesicle recycling, and shi(ts1) is semidominant, a simple temperature shift should lead to fast and reversible effects on synaptic transmission of shi(ts1) expressing neurons. When shi(ts1) expression was directed to cholinergic neurons, adult flies showed a dramatic response to the restrictive temperature, becoming motionless within 2 min at 30 degrees C. This temperature-induced paralysis was reversible. After being shifted back to the permissive temperature, they readily regained their activity and started to walk in 1 min. When shi(ts1) was expressed in photoreceptor cells, adults and larvae exhibited temperature-dependent blindness. These observations show that the GAL4/UAS system can be used to express shi(ts1) in a specific subset of neurons to cause temperature-dependent changes in behavior. Because this method allows perturbation of the neuronal activities rapidly and reversibly in a spatially and temporally restricted manner, it will be useful to study the functional significance of particular neuronal subsets in the behavior of intact animals.     

Sensitization of cardiac vagal afferent reflexes at the sensory receptor level: an overview

The gain or sensitivity of reflexes originating in cardiac sensory receptors with vagal afferent pathways is highly dynamic. This modulation is usually attributed to central nervous system or efferent mechanisms. This paper briefly reviews evidence that modulation of reflexes originating in the heart can also occur at the sensory or afferent level. Five examples are cited: calcium antagonists, cardiac glycosides, arginine vasopressin, atrial natriuretic peptides, and changes in dietary sodium. These examples emphasize the role of ionic and humoral factors in regulation of cardiac vagal afferent function. This concept of sensory modulation of cardiac vagal afferents has implications for cardiovascular pharmacology and for pathophysiological states such as heart failure and hypertension.     

Angiotensin II potentiates adrenergic and muscarinic modulation of guinea pig intracardiac neurons

The intrinsic cardiac plexus represents a major peripheral integration site for neuronal, hormonal, and locally produced neuromodulators controlling efferent neuronal output to the heart. This study examined the interdependence of norepinephrine, muscarinic agonists, and ANG II, to modulate intrinsic cardiac neuronal activity. Intracellular voltage recordings from whole-mount preparations of the guinea pig cardiac plexus were used to determine changes in active and passive electrical properties of individual intrinsic cardiac neurons. Application of either adrenergic or muscarinic agonists induced changes in neuronal resting membrane potentials, decreased afterhyperpolarization duration of single action potentials, and increased neuronal excitability. Adrenergic responses were inhibited by removal of extracellular calcium ions, while muscarinic responses were inhibited by application of TEA. The adrenergic responses were heterogeneous, responding to a variety of receptor-specific agonists (phenylephrine, clonidine, dobutamine, and terbutaline), although α-receptor agonists produced the most frequent responses. Application of ANG II alone produced a significant increase in excitability, while application of ANG II in combination with either adrenergic or muscarinic agonists produced a much larger potentiation of excitability. The ANG II-induced modulation of firing was blocked by the angiotensin type 2 (AT(2)) receptor inhibitor PD 123319 and was mimicked by the AT(2) receptor agonist CGP-42112A. AT(1) receptor blockade with telmasartin did not alter neuronal responses to ANG II. These data demonstrate that ANG II potentiates both muscarinically and adrenergically mediated activation of intrinsic cardiac neurons, doing so primarily via AT(2) receptor-dependent mechanisms. These neurohumoral interactions may be fundamental to regulation of neuronal excitability within the intrinsic cardiac nervous system.