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We reported pharmacological data suggesting that stimulation of a vago-vagal reflex activates GABAergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the antrum, but not to the fundus (Ferreira et al., 2002). The purpose of this study was to use an ultrastructural approach to test the hypothesis that GABAergic terminals form synapses with DMV antrum-projecting neurons, but not with DMV fundus-projecting neurons. A retrograde tracer, CTB-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of anesthetized rats. Animals were re-anesthetized 48 h later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTB-HRP, and immunocytochemically for glutamic acid decarboxylase isoenzyme 67 immunoreactivity (GAD67-IR) by dual-labeling electron microscopic methods. Most cell bodies and dendrites of neurons that were retrogradely labeled from the stomach occurred at the level of the area postrema. Examination of 214 synapses on 195 neurons that projected to the antrum revealed that 23.0+/-3.6% (n = 4) of synaptic contacts were with GAD67-IR terminals. The examination of 220 synapses on 203 fundus-projecting neurons revealed that only 7.9+/-3.1% (n = 4) of synaptic contacts were with GAD67-IR terminals. The difference between GAD67-IR synaptic contacts with antrum- and fundus-projecting neurons was statistically significant (p<0.05). These data suggest that brainstem circuitry controlling the antrum involves GABAergic transmission., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We reported pharmacological data suggesting that stimulation of the vago-vagal reflex activates noradrenergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the fundus, but not to the antrum [Ferreira Jr., M., Sahibzada, N., Shi, M., Panico, W., Neidringhaus, M., Wasserman, A., Kellar, K.J., Verbalis, J., Gillis, R.A., 2002. CNS site of action and brainstem circuitry responsible for the intravenous effects of nicotine on gastric tone. J. Neurosci. 22, 2764-2779.]. The purpose of this study was to use an ultrastructural approach to test the hypothesis that noradrenergic terminals form synapses with DMV fundus-projecting neurons, but not with DMV antrum-projecting neurons. A retrograde tracer, CTbeta-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of rats. Animals were re-anesthetized 48 h later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTbeta-HRP, and immunocytochemically for either DbetaH or PNMT by dual-labeling electron microscopic methods. Most cell bodies and dendrites of neurons that were retrogradely labeled from the stomach occurred at the level of the area postrema. Examination of 482 synapses on 238 neurons that projected to the fundus revealed that 17.4+/-2.7% (n=4) of synaptic contacts were with DbetaH-IR terminals. Of 165 fundus-projecting neurons, 4.4+/-1.5% (n=4) formed synaptic contacts with PNMT-IR terminals. In contrast, the examination of 384 synapses on 223 antrum-projecting neurons revealed no synaptic contact with DbetaH-IR terminals. These data provide proof that norepinephrine containing nerve terminals synapse with DMV fundus-projecting neurons but not with DMV antrum-projecting neurons. These data also suggest that brainstem circuitry controlling the fundus differs from circuitry controlling the antrum.

In cat, distinct populations of vagal preganglionic and postganglionic neurons selectively modulate heart rate, atrioventricular conduction and left ventricular contractility, respectively. Vagal preganglionic neurons to the heart originate in the ventrolateral part of nucleus ambiguus and project to postganglionic neurons in intracardiac ganglia, including the sinoatrial (SA), atrioventricular (AV) and cranioventricular (CV) ganglia, which selectively modulate heart rate, AV conduction and left ventricular contractility, respectively. These ganglia receive projections from separate populations of vagal preganglionic neurons. The neurochemical anatomy and synaptic interactions of afferent neurons which mediate central control of these preganglionic neurons is incompletely understood. Enkephalins cause bradycardia when microinjected into nucleus ambiguus. It is not known if this effect is mediated by direct synapses of enkephalinergic terminals upon vagal preganglionic neurons to the heart. The effects of opioids in nucleus ambiguus upon AV conduction and cardiac contractility have also not been studied. We have tested the hypothesis that enkephalinergic nerve terminals synapse upon vagal preganglionic neurons projecting to the SA, AV and CV ganglia. Electron microscopy was used combining retrograde labeling from the SA, AV or CV ganglion with immunocytochemistry for enkephalins in ventrolateral nucleus ambiguus. Eight percent of axodendritic synapses upon negative chronotropic, and 12% of axodendritic synapses upon negative dromotropic vagal preganglionic neurons were enkephalinergic. Enkephalinergic axodendritic synapses were also present upon negative inotropic vagal preganglionic neurons. Thus enkephalinergic terminals in ventrolateral nucleus ambiguus can modulate not only heart rate but also atrioventricular conduction and left ventricular contractility by directly synapsing upon cardioinhibitory vagal preganglionic neurons.

This review summarizes recent work on two basic processes of central nervous system (CNS) control of cholinergic outflow to the airways: 1) transmission of bronchoconstrictive signals from the airways to the airway-related vagal preganglionic neurons (AVPNs) and 2) regulation of AVPN responses to excitatory inputs by central GABAergic inhibitory pathways. In addition, the autocrine-paracrine modulation of AVPNs is briefly discussed. CNS influences on the tracheobronchopulmonary system are transmitted via AVPNs, whose discharge depends on the balance between excitatory and inhibitory impulses that they receive. Alterations in this equilibrium may lead to dramatic functional changes. Recent findings indicate that excitatory signals arising from bronchopulmonary afferents and/or the peripheral chemosensory system activate second-order neurons within the nucleus of the solitary tract (NTS), via a glutamate-AMPA signaling pathway. These neurons, using the same neurotransmitter-receptor unit, transmit information to the AVPNs, which in turn convey the central command to airway effector organs: smooth muscle, submucosal secretory glands, and the vasculature, through intramural ganglionic neurons. The strength and duration of reflex-induced bronchoconstriction is modulated by GABAergic-inhibitory inputs and autocrine-paracrine controlling mechanisms. Downregulation of GABAergic inhibitory influences may result in a shift from inhibitory to excitatory drive that may lead to increased excitability of AVPNs, heightened airway responsiveness, and sustained narrowing of the airways. Hence a better understanding of these normal and altered central neural circuits and mechanisms could potentially improve the design of therapeutic interventions and the treatment of airway obstructive diseases.

The vagal postganglionic control of cardiac rate is mediated by two intracardiac ganglia, i.e., the sinoatrial (SA) and posterior atrial (PA) ganglia. Nothing is known about the vagal preganglionic neurons (VPNs) that innervate the PA ganglion or about the neurochemical anatomy of central afferents that innervate these VPNs. These issues were examined using light microscopic retrograde labeling methods and dual-labeling electron microscopic histochemical and immunocytochemical methods. VPNs projecting to the PA ganglion are found in a narrow column exclusively in the ventrolateral nucleus ambiguus (NA-VL). These neurons are relatively large (37.6 +/- 2.7 microm by 21.3 +/- 3.4 microm) with abundant cytoplasm and intracellular organelles, rare somatic and dendritic spines, round uninvaginated nuclei, and myelinated axons. Previous physiological data indicated that microinjections of neuropeptide Y (NPY) into the NA-VL cause negative chronotropic effects. The present morphological data demonstrate that NPY-immunoreactive nerve terminals formed 18 +/- 4% of the axodendritic or axosomatic synapses and close appositions on VPNs projecting to the PA ganglion. Three approximately equal populations of VPNs in the NA-VL were retrogradely labeled from the SA and PA ganglia. One population each projects to the SA ganglion, the PA ganglion, or to both the SA and PA ganglia. Therefore, there are both shared and independent pathways involved in the vagal preganglionic controls of cardiac rate. These data are consistent with the hypothesis that the central and peripheral parasympathetic controls of cardiac rate are coordinated by multiple potentially redundant and/or interacting pathways and mechanisms.

Intracardiac pathways mediating the parasympathetic control of various cardiac functions are incompletely understood. Several intracardiac ganglia have been demonstrated to potently influence cardiac rate [the sinoatrial (SA) ganglion], atrioventricular (AV) conduction (the AV ganglion), or left ventricular contractility (the cranioventricular ganglion). However, there are numerous ganglia found throughout the heart whose functions are poorly characterized. One such ganglion, the posterior atrial (PA) ganglion, is found in a fat pad on the rostral dorsal surface of the right atrium. We have investigated the potential impact of this ganglion on cardiac rate and AV conduction. We report that microinjections of a ganglionic blocker into the PA ganglion significantly attenuates the negative chronotropic effects of vagal stimulation without significantly influencing negative dromotropic effects. Because prior evidence indicates that the PA ganglion does not project to the SA node, we neuroanatomically tested the hypothesis that the PA ganglion mediates its effect on cardiac rate through an interganglionic projection to the SA ganglion. Subsequent to microinjections of the retrograde tracer fast blue into the SA ganglion, >70% of the retrogradely labeled neurons found within five intracardiac ganglia throughout the heart were observed in the PA ganglion. The neuroanatomic data further indicate that intraganglionic neuronal circuits are found within the SA ganglion. The present data support the hypothesis that two interacting cardiac centers, i.e., the SA and PA ganglia, mediate the peripheral parasympathetic control of cardiac rate. These data further support the emerging concept of an intrinsic cardiac nervous system.

The locations, projections, and functions of the intracardiac ganglia are incompletely understood. Immunocytochemical labeling with the general neuronal marker protein gene product 9.5 (PGP 9.5) was used to determine the distribution of intracardiac neurons throughout the cat atria and ventricles. Fluorescence microscopy was used to determine the number of neurons within these ganglia. There are eight regions of the cat heart that contain intracardiac ganglia. The numbers of neurons found within these intracardiac ganglia vary dramatically. The total number of neurons found in the heart (6,274 +/- 1,061) is almost evenly divided between the atria and the ventricles. The largest ganglion is found in the interventricular septum (IVS). Retrogradely labeled fluorescent tracer studies indicated that the vagal intracardiac innervation of the anterior surface of the right ventricle originates predominantly in the IVS ganglion. A cranioventricular (CV) ganglion was retrogradely labeled from the anterior surface of the left ventricle but not from the anterior surface of the right ventricle. These new neuroanatomic data support the prior physiological hypothesis that the CV ganglion in the cat exerts a negative inotropic effect on the left ventricle. A total of three separate intracardiac ganglia innervate the left ventricle, i.e., the CV, IVS, and a second left ventricular (LV2) ganglion. However, the IVS ganglion provides the major source of innervation to both the left and right ventricles. This dual innervation pattern may help to coordinate or segregate vagal effects on left and right ventricular performance.

GABA is the main inhibitory neurotransmitter that participates in the regulation of cholinergic outflow to the airways. We have tested the hypothesis that a monosynaptic GABAergic circuit modulates the output of airway-related vagal preganglionic neurons (AVPNs) in the rostral nucleus ambiguus by using a dual-labeling electron microscopic method combining immunocytochemistry for glutamic acid decarboxylase (GAD) with retrograde tracing from the trachea. We also determined the effects of blockade of GABAA receptors on airway smooth muscle tone. The results showed that retrogradely labeled AVPNs received a significant GAD-immunoreactive (GAD-IR) terminal input. Out of a pooled total of 3,161 synaptic contacts with retrogradely labeled somatic and dendritic profiles, 20.2% were GAD-IR. GAD-IR terminals formed significantly more axosomatic synapses than axodendritic synapses (P < 0.02). A dense population of GABAergic synaptic contacts on AVPNs provides a morphological basis for potent physiological effects of GABA on the excitability of AVPNs. GAD-IR terminals formed exclusively symmetric synaptic specializations. GAD-IR terminals were significantly larger (P < 0.05) in both length and width than unlabeled terminals synapsing on AVPNs. Therefore, the structural characteristics of certain nerve terminals may be closely correlated with their function. Pharmacological blockade of GABAA receptors within the rostral nucleus ambiguus increased activity of putative AVPNs and airway smooth muscle tone. We conclude that a tonically active monosynaptic GABAergic circuit utilizing symmetric synapses regulates the discharge of AVPNs.

In this study, we have investigated the ultrastructure and function of the catecholaminergic circuitry modulating the output of airway-related vagal preganglionic neurons (AVPNs) in ferrets. Immunoelectron microscopy was employed to characterize the nature of catecholaminergic innervation of AVPN at the ultrastructural level. In addition, immunofluorescence was used to examine the expression of the alpha(2A)-adrenergic receptor (alpha(2A)-AR) on AVPNs, and norepinephrine release within the rostral nucleus ambiguous (rNA) was measured by using microdialysis. Physiological experiments were performed to determine the effects of stimulation of the noradrenergic locus coeruleus (LC) cell group on airway smooth muscle tone. The results showed that 1) catecholaminergic nerve endings terminate in the vicinity of identified AVPNs but very rarely form axosomatic or axodendritic synapses with the AVPNs that innervate the extrathoracic trachea; 2) AVPNs express the alpha(2A)-AR; 3) LC stimulation-induced norepinephrine release within the rNA region was associated with airway smooth muscle relaxation; and 4) blockade of alpha(2A)-AR on AVPNs diminished the inhibitory effects of LC stimulation on airway smooth muscle tone. It is concluded that a noradrenergic circuit originating within the LC is involved in the regulation of AVPN activity within the rNA, and stimulation of the LC dilates the airways by the release of norepinephrine and activation of alpha(2A)-AR expressed by AVPNs, mainly via volume transmission.

Airway disorders, such as asthma and chronic obstructive bronchitis, are, in part, due to abnormalities in the nervous control of the airways. However, the ultrastructural circuitry and neurochemical anatomy of afferents modulating the output of airway-related vagal preganglionic neurons (VPNs) in the nucleus ambiguus are poorly understood. We have examined the potential role of substance P (SP) immunoreactive afferents in the regulation of anatomically identified airway VPNs. Cholera toxin b-subunit conjugated to horseradish peroxidase was used as a retrograde cell body tracer to identify the central VPNs innervating the extra-thoracic trachea. Immunocytochemistry was employed to identify SP afferents. The external formation of the nucleus ambiguus was examined by electron microscopy using a simultaneous double labeling method. Cell bodies of tracheal VPNs were 31.7 +/- 1.18 x 23.0 +/- 1.3 microm (means +/- S.E.M.) in size, contained abundant endoplasmic reticulum, had a round nucleus with a prominent nucleolus, no satellite body and displayed somatic and dendritic spines. Somato-somatic appositions, somato-dendritic appositions without intervening glial processes and dendritic "bundling" commonly seen in esophageal motoneurons were not observed. The ultrastructural morphology of tracheal VPNs were also clearly distinguishable from pharyngeal and laryngeal motoneurons in other divisions of the nucleus ambiguus which lack somatic spines. These data are consistent with the hypothesis that differences in the ultrastructure and synaptology of the different divisions of the nucleus ambiguus may be associated with specific physiological functions. The mean size (+/- S.E.M.) of SP nerve terminals was 1.57 +/- 0.06 x 0.79 +/- 0.03 microm. SP terminals formed 17.5% of the axo-dendritic and 15.9% of the axo-somatic synapses which were observed upon retrogradely labeled tracheal VPNs. Synaptic contacts observed were both symmetric and asymmetric. These synaptic interactions define, in part, the neurochemical anatomy of neuronal circuits modulating vagal preganglionic control of tracheal functions.

Previous research from this laboratory has shown that substance P-immunoreactive (SP) terminals synapse upon negative chronotropic vagal preganglionic neurons (VPNs), but not upon negative dromotropic VPNs, of the ventrolateral nucleus ambiguus (NA-VL). Moreover, SP agonists injected into NA-VL cause bradycardia without decreasing AV conduction. In the current study, we have: (1) defined the electron microscopic characteristics of the SP neurons of NA-VL in dog; and (2) tested the hypothesis that SP nerve terminals synapse upon negative inotropic VPNs of NA-VL, retrogradely labeled from the cranial medial ventricular (CMV) ganglion. Numerous SP terminals and a few SP neurons were observed in the vicinity of retrogradely labeled neurons. SP terminals were observed forming synapses with unlabeled dendrites and with SP dendrites, but never with the retrogradely labeled neurons. Together, these results and earlier findings suggest that SP agonists may be able to induce bradycardia without decreasing AV conduction or ventricular contractility., (Copyright 1998 Elsevier Science B.V.)

Recent physiological evidence indicates that vagal postganglionic control of left ventricular contractility is mediated by neurons found in a ventricular epicardial fat pad ganglion. In the dog this region has been referred to as the cranial medial ventricular (CMV) ganglion [J.L. Ardell, Structure and function of mammalian intrinsic cardiac neurons, in: J.A. Armour, J.L. Ardell (Eds.). Neurocardiology, Oxford Univ. Press, New York, 1994, pp. 95-114; B.X. Yuan, J.L. Ardell, D.A. Hopkins, A.M. Losier, J.A. Armour, Gross and microscopic anatomy of the canine intrinsic cardiac nervous system, Anat. Rec., 239 (1994) 75-87]. Since activation of the vagal neuronal input to the CMV ganglion reduces left ventricular contractility without influencing cardiac rate or AV conduction, this ganglion contains a functionally selective pool of negative inotropic parasympathetic postganglionic neurons. In the present report we have defined the light microscopic distribution of preganglionic negative inotropic neurons in the CNS which are retrogradely labeled from the CMV ganglion. Some tissues were also processed for the simultaneous immunocytochemical visualization of tyrosine hydroxylase (TH: a marker for catecholaminergic neurons) and examined with both light microscopic and electron microscopic methods. Histochemically visualized neurons were observed in a long slender column in the ventrolateral nucleus ambiguus (NA-VL). The greatest number of retrogradely labeled neurons were observed just rostral to the level of the area postrema. TH perikarya and dendrites were commonly observed interspersed with vagal motoneurons in the NA-VL. TH nerve terminals formed axo-dendritic synapses upon negative inotropic vagal motoneurons, however the origin of these terminals remains to be determined. We conclude that synaptic interactions exist which would permit the parasympathetic preganglionic vagal control of left ventricular contractility to be modulated monosynaptically by catecholaminergic afferents to the NA-VL.

We hypothesized that selective control of ventricular contractility might be mediated by postganglionic parasympathetic neurons in the cranial medial ventricular (CMV) ganglion plexus located in a fat pad at the base of the aorta. Sinus rate, atrioventricular (AV) conduction (ventricular rate during atrial pacing), and left ventricular contractile force (LV dP/dt during right ventricular pacing) were measured in eight chloralose-anesthetized dogs both before and during bilateral cervical vagus stimulation (20-30 V, 0.5 ms pulses, 15-20 Hz). Seven of these dogs were tested under beta-adrenergic blockade (propranolol, 0.8 mg kg(-1) i.v.). Control responses included sinus node bradycardia or arrest during spontaneous rhythm, high grade AV block or complete heart block, and a 30% decrease in contractility from 2118 +/- 186 to 1526 +/- 187 mm Hg s(-1) (P < 0.05). Next, the ganglionic blocker trimethaphan (0.3-1.0 ml of a 50 microg ml(-1) solution) was injected into the CMV fat pad. Then vagal stimulation was repeated, which now produced a relatively small 5% (N.S., P > 0.05) decrease in contractility but still elicited the same degree of sinus bradycardia and AV block (N = 8, P < 0.05). Five dogs were re-tested 3 h after trimethaphan fat pad injection, at which time blockade of vagally-induced negative inotropy was partially reversed, as vagal stimulation decreased LV dP/dt by 19%. The same dose of trimethaphan given either locally into other fat pads (PVFP or IVC-ILA) or systemically (i.v.) had no effect on vagally-induced negative inotropy. Thus, parasympathetic ganglia located in the CMV fat pad mediated a decrease in ventricular contractility during vagal stimulation. Blockade of the CMV fat pad had no effect on vagally-mediated slowing of sinus rate or AV conduction.

Physiological and light microscopic evidence suggest that substance P (SP) may be a neurotransmitter contained in first-order sensory baroreceptor afferents; however, ultrastructural support for this hypothesis is lacking. We have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase (HRP). The dorsolateral subnucleus of the nucleus tractus solitarius (dlNTS) was processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical visualization of SP by dual labeling light and electron microscopic methods. Either HRP or SP was readily identified in single-labeled unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the dlNTS, which were simultaneously identified as CSN primary afferents. However, only 15% of CSN terminals in the dlNTS were immunoreactive for SP. Therefore, while the ultrastructural data support the hypothesis that SP immunoreactive first-order neurons are involved in the origination of the baroreceptor reflex, they suggest that only a modest part of the total sensory input conveyed from the carotid sinus baroreceptors to the dlNTS is mediated by SP immunoreactive CSN terminals. Five types of axo-axonic synapses were observed in the dlNTS. SP immunoreactive CSN afferents were very rarely involved in these synapses. Furthermore, SP terminals were never observed to form the presynaptic element in an axo-axonic synapse with a CSN afferent. Therefore, SP does not appear to be involved in the modulation of the baroreceptor reflex in the dlNTS., (Copyright 1998 Elsevier Science B.V.)

We have tested the hypothesis: (1) that presumptive negative dromotropic vagal preganglionic neurons in the ventrolateral nucleus ambiguus (NA-VL) can be selectively labelled from the heart, by injecting one of two fluorescent tracers into the two intracardiac ganglia which independently control sino-atrial (SA) rate or atrioventricular (AV) conduction; i.e., the SA and AV ganglia, respectively. The NA-VL was examined for the presence of single and/or double labelled cells. Over 91% of vagal preganglionic neurons in the NA-VL projecting to either intracardiac ganglion did not project to the second ganglion. Consequently, we also tested the hypothesis: (2) that there is a monosynaptic connection between neurons of the medial, and/or dorsolateral nucleus of the solitary tract (NTS), rostral to obex, and negative dromotropic neurons in the NA-VL. An anterograde tracer was injected into the NTS, and a retrograde tracer into the AV ganglion. The anterograde marker was found in both myelinated and unmyelinated axons in the NA-VL, as well as in nerve terminals. Axo-somatic and axo-dendritic synapses were detected between terminals labelled from the NTS, and retrogradely labelled negative dromotropic neurons in the NA-VL. This is the first ultrastructural demonstration of a monosynaptic pathway between neurons in the NTS and functionally associated (negative dromotropic) cardioinhibitory neurons. The data are consistent with the hypothesis that the neuroanatomical circuitry mediating the vagal baroreflex control of AV conduction may be composed of as few as four neurons in series, although interneurons may also be interposed within the NTS., (Copyright 1998 Elsevier Science B.V.)

Activation of the vagus nerve leads to decreases in sinoatrial (SA) rate, atrioventricular (AV) conduction, and myocardial contractility. Previous data are consistent with the hypothesis that vagal control of cardiac rate and AV conduction are mediated by two anatomically separated and physiologically independent parasympathetic intracardiac ganglia located in fat pads on the surface of the right and left atria, respectively. These data suggested that vagal control of ventricular contractility might be mediated through another intracardiac ganglion. We examined the ventricles of cat hearts histologically for the presence of ganglia. Multiple small basophilic ganglia composed of a few neurons, and an occasional larger ganglion were found embedded in the epicardial fat surrounding the cranial margin of the anterior surface of the left ventricle, near the juncture with the right ventricle, which we refer to as the CV ganglion. In anesthetized cats, right cervical vagal stimulation decreased SA rate by 44 +/- 5%, decreased the rate of AV conduction by 68 +/- 14%, and reduced ventricular contractility by 19.5 +/- 5.7%. Vagally induced negative inotropism was almost completely prevented by microinjection of a ganglionic blocking drug into the CV ganglion. However, these injections into the CV ganglion did not significantly effect vagally induced decreases in either SA rate or AV conduction. We conclude: (1) that ganglia are found in a fat pad on the surface of the left ventricle of the cat heart and (2) that the CV ganglion selectively mediates the negative inotropic effect of vagal stimulation on the left ventricle. Greater understanding of the physiological functions of intracardiac neuronal circuits may help in developing new strategies to treat disorders of cardiac contractility such as congestive heart failure.

This study has investigated the synaptic interactions between hypoglossal motoneurons and substance P (SP)-immunoreactive terminals. Cholera toxin B conjugated to horseradish peroxidase was injected into the tip of the tongue on the right side of six ketamine-anesthetized cats. Two to five days later, the animals were killed. Cells containing HRP were labeled with a histochemical reaction utilizing tetramethylbenzidine (TMB) as the chromogen. TMB forms crystalline reaction products that are very distinct at the electron microscopic level. The tissues were then processed for immunocytochemistry using an antiserum against SP. The chromogen used in this case, diaminobenzidine, yields amorphous reaction products. At the light microscopic level, labeled cells were observed primarily ipsilaterally in both intermediate and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were seen at the level of obex. At the electron microscopic level, both asymmetric and symmetric synapses were observed. SP-immunoreactive nerve terminals formed asymmetric synapses with labeled dendrites and symmetric synapses with labeled perikarya. SP-labeled terminals also synapsed on unlabeled dendrites and somata. These are the first ultrastructural studies demonstrating synaptic interactions between hypoglossal motoneurons and SP terminals. These studies demonstrate that hypoglossal motoneurons that innervate intrinsic tongue muscles are modulated by SP and that SP may play a role in the control of fine movements of the tongue.

Recent data indicate that there is a cardiotopic organization of negative chronotropic and negative dromotropic neurons in the nucleus ambiguus (NA). Negative dromotropic neurons are found in the rostral ventrolateral NA (rNA-VL), negative chronotropic neurons are found in the caudal ventrolateral NA (cNA-VL), and both types of neurons are found in an intermediate level of the ventrolateral NA (iNA-VL). Substance P (SP) immunoreactive nerve terminals synapse upon negative chronotropic vagal motoneurons in the iNA-VL, and SP microinjections in the NA cause bradycardia. In the present report we have attempted to: (1) define the type of tachykinin receptor which mediates the negative chronotropic effect of SP microinjections into the iNA-VL; (2) define the physiological effect of microinjections of a selective SP agonist into the rNA-VL on atrioventricular (AV) conduction: and (3) find ultrastructural evidence for synaptic interactions of SP-immunoreactive nerve terminals with negative dromotropic vagal motoneurons in the rNA-VL. Microinjections of the excitatory amino acid glutamate (Glu) into the iNA-VL to activate all local vagal preganglionic neurons caused both bradycardia and a decrease in the rate of AV conduction. Injections of the selective neurokinin-1 (NK-1) receptor agonist drug GR-73632 also caused bradycardia, however the rapid onset of agonist induced desensitization prevented an evaluation of potential effects on AV conduction in the iNA-VL. These data suggest that the SP-induced bradycardia which can be elicited from the NA is mediated, at least in part, by NK-1 receptors. Microinjections of Glu into the rNA-VL caused a decrease in AV conduction without an effect on cardiac rate. On the other hand, GR-73632 microinjections into rNA-VL did not affect AV conduction. Following injections of the beta subunit of cholera toxin conjugated to horseradish peroxidase (CTB-HRP) into the left atrial fat pad ganglion which selectively mediates changes in AV conduction, retrogradely labeled neurons were histochemically visualized in the rNA-VL. These tissues were subsequently processed for the simultaneous immunocytochemical visualization of SP, and examined by electron microscopy. Histochemically labeled neurons were large, multipolar, with abundant cytoplasm containing large masses of rough endoplasmic reticulum, and exhibited distinctive dendritic and somatic spines. Unlabeled nerve terminals were noted to form either asymmetric or symmetric synapses with dendrites, dendritic spines, and perikarya of histochemically labeled neurons. SP-immunoreactive nerve terminals were also detected in the rNA-VL. SP terminals typically contained numerous small pleomorphic vesicles, multiple large dense core vesicles, and several mitochondria, and they synapsed upon unlabeled dendritic profiles. A total of 154 SP-immunoreactive nerve terminals were observed on photomicrographs of tissues which also contained histochemically labeled profiles. None made an identifiable synapse with a retrogradely labeled profile on the sections examined. In summary, both physiological and ultrastructural data indicate that SP terminals in the iNA-VL do modify the output of negative chronotropic vagal motoneurons. This effect is mediated by NK-1 receptors. On the other hand both physiological and ultrastructural data indicate that SP terminals in the rNA-VL do not modify the output of negative dromotropic vagal motoneurons. Therefore different mechanisms (neurotransmitters or receptors) mediate the central vagal control of cardiac rate and AV conduction.

Tyrosine hydroxylase immunoreactive sensory neurons in the petrosal ganglion selectively innervate the carotid body via the carotid sinus nerve. Central projections of the carotid sinus nerve were traced with horseradish peroxidase. The commissural nucleus of the tractus solitarius was examined by dual labelling light and electron microscopy. Dense bilateral labelling with horseradish peroxidase was found in the tractus solitarius and commissural nucleus of the tractus solitarius. Horseradish peroxidase was found in unmyelinated axons, myelinated axons, and nerve terminals. About 88% of horseradish peroxidase-labelled carotid sinus nerve axons were unmyelinated. Tyrosine hydroxylase immunoreactivity was identified in unmyelinated axons, myelinated axons, dendrites, perikarya, and nerve terminals. Most tyrosine hydroxylase immunoreactive axons (93%) in the commissural nucleus of the tractus solitarius were unmyelinated. Tyrosine hydroxylase immunoreactivity was simultaneously identified in carotid sinus nerve unmyelinated axons, myelinated axons, and nerve terminals. These double-labelled terminals comprised 28% of the number of tyrosine hydroxylase immunoreactive terminals in the commissural nucleus of the tractus solitarius, and 55% of transganglionically-labelled terminals. Therefore, there are both central and peripheral sources of tyrosine hydroxylase immunoreactive nerve terminals in the commissural nucleus of the tractus solitarius. These data support the hypothesis that peripheral tyrosine hydroxylase immunoreactive neurons are involved in the origination of the chemoreceptor reflex. Axo-axonic synapses between peripheral carotid sinus nerve afferent terminals and central terminals containing tyrosine hydroxylase immunoreactivity were observed in 22% of the axo-axonic synapses observed. Thus, central tyrosine hydroxylase immunoreactivity neurons are involved in the modulation of the chemo-and/or baroreceptor reflexes. Synaptic contacts were not observed between carotid sinus nerve afferents and tyrosine hydroxylase immunoreactive perikarya of dendrites. Catecholaminergic neurons are thus unlikely to be the second order neurons of either the chemo-or baroreceptor reflex in the commissural nucleus of the tractus solitarius.

Previous anatomic data have described the distribution of presumptive negative chronotropic and negative dromotropic neurons in the ventro-lateral nucleus ambiguus (NA-VL) following injections of retrograde tracers into physiologically selective parasympathetic intracardiac ganglia. Negative dromotropic neurons were preferentially distributed in the rostral NA-VL (rNA-VL). Negative chronotropic neurons were preferentially distributed in the caudal NA-VL (cNA-VL). Significant numbers of both types of cardio-inhibitory neurons were observed to overlap in an intermediate level of the NA-VL (iNA-VL). In the present report, we have examined the effects of microinjections of the excitatory amino-acid glutamate (GLU) into the cNA-VL and iNA-VL on cardiac rate and AV conduction while recording the electrocardiogram in paced and non-paced cat hearts. The data indicate that: (i) excitation of neurons in the cNA-VL causes a 58 +/- 17% reduction in cardiac rate, without influencing AV conduction; and (ii) excitation of neurons in the iNA-VL causes both a reduction in heart rate (68 +/- 12%) and a decrease in the rate of AV conduction (38 +/- 7%). These physiological results support the anatomical inference that neurons in the cNA-VL that are retrogradely labeled from physiologically selective parasympathetic intracardiac ganglia selectively exhibit negative chronotropic properties. Furthermore, the data indicate that there is a longitudinal cardiotopic organization of both negative chronotropic and negative dromotropic neurons in the NA-VL. This CNS organization mirrors the peripheral organization of functionally selective cardiac components of the vagus nerve. Finally, the data are consistent with the hypothesis that anatomically separated and functionally selective parasympathetic preganglionic vagal motoneurons in the NA independently control cardiac rate and AV conduction.

The neurochemical anatomy and synaptic interactions of morphologically identified chemoreceptor or baroreceptor afferents in the nucleus of the solitary tract (NTS) are poorly understood. A substantial body of physiological and light microscopic evidence suggests that substance P (SP) may be a neurotransmitter contained in first order sensory chemo- or baroreceptor afferents, however ultrastructural support of this hypothesis is lacking. In the present report we have traced the central projections of the carotid sinus nerve (CSN) in the cat by utilizing the transganglionic transport of horseradish peroxidase. Medullary tissues including the commissural NTS (cNTS) were processed for the histochemical visualization of transganglionically labeled CSN afferents and for the immunocytochemical detection of SP by dual labeling light and electron microscopic methods. At the light microscopic level, dense bilateral labeling with TMB was found in the tractus solitarius (TS) and cNTS, caudal to the obex. Rostral to the obex, significant ipsilateral TMB labeling was detected in the dorsal, dorso-lateral, and medial subnuclei of the NTS, as well as in the TS. Significant staining of SP immunoreactive processes was detected in most subnuclei of the NTS. The cNTS was examined by electron microscopy. Either HRP or SP were readily identified in single labeled unmyelinated axons, myelinated axons, and nerve terminals in the cNTS. SP immunoreactivity was also identified in unmyelinated axons, myelinated axons, and nerve terminals in the cNTS which were simultaneously identified as CSN primary afferents. These ultrastructural data support the hypothesis that SP immunoreactive first order neurons are involved in the origination of the chemo- and baroreceptor reflexes. Axo-axonic synapses were observed between CSN primary afferent terminals and: (a) unlabeled nerve terminals; (b) other CSN primary afferent terminals; and (c) terminals containing SP. Axo-axonic synapses were also observed between CSN primary afferents which contained SP, and other SP terminals. These observations may mediate the morphological bases for multiple forms of presynaptic inhibition in the cNTS, including those involved in cardiorespiratory integration. In conclusion, our results indicate that SP immunoreactive nerve terminals may be important in both the origination and the modulation of the chemo- and/or baroreceptor reflexes.

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic postganglionic vagal motoneurons on the surface of the heart which are capable of selective control of sinoatrial rate, atrioventricular conduction and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates atrioventricular conduction (the AV ganglion). Medullary tissues were processed for the histochemical detection of retrogradely labeled neurons by light and electron microscopic methods. Negative dromotropic retrogradely labeled cells were found in a long column in the ventrolateral nucleus ambiguus (NA-VL), which enlarged somewhat at the level of the area postrema, but reached its largest size rostral to the area postrema in an area termed the rostral ventrolateral nucleus ambiguus (rNA-VL). Three times as many cells were observed in the left rNA-VL as compared to the right (P < 0.025). Retrogradely labeled cells were also consistantly observed in the dorsal motor nucleus of the vagus (DMV). The DMV contained one third as many cells as the NA-VL. The right DMV contained twice as many cells as the left (P < 0.05). These data are consistent with physiological evidence that suggests that the left vagus nerve is dominant in the regulation of AV conduction, but that the right vagus nerve is also influential. While recording the electrocardiogram in paced and non-paced hearts, L-glutamate (GLU) was microinjected into the rNA-VL. Microinjections of GLU caused a 76% decrease in the rate of atrioventricular (AV) conduction (P < 0.05) and occasional second degree heart block, without changing heart rate. The effects of GLU were abolished by ipsilateral cervical vagotomy. These physiological data therefore support the anatomical inference that CNS neurons that are retrogradely labeled from the AV ganglion selectively exhibit negative dromotropic properties. Retrogradely labeled negative dromotropic neurons displayed a round nucleus with ample cytoplasm, abundant rough endoplasmic reticulum and the presence of distinctive somatic and dendritic spines. These neurons received synapses from afferent terminals containing small pleomorphic vesicles and large dense core vesicles. These terminals made both asymmetric and symmetric contacts with negative dromotropic dendrites and perikarya, respectively. In conclusion, the data presented indicate that there is a cardiotopic organization of ultrastructurally distinctive negative dromotropic neurons in the NA-VL. This central organization of parasympathetic preganglionic vagal motoneurons mirrors the functional organization of cardioinhibitory postganglionic neurons of the peripheral vagus nerve. These data are further discussed in comparison to a recent report on the light microscopic distribution and ultrastructural characteristics of negative chronotropic neurons in the NA-VL42.(ABSTRACT TRUNCATED AT 400 WORDS)

Experiments utilizing surgical parasympathectomy of discrete fat pad ganglia on the surface of the heart have suggested that there are two anatomically segregated and physiologically independent parasympathetic intracardiac ganglia which are capable of selective control of sino-atrial (SA) rate and atrio-ventricular (AV) conduction. Some pharmacological data, however, are inconsistent with these conclusions. We have examined the cardiodynamic effects of discrete injections of a ganglionic blocking drug into two fat pads on the surface of the cat heart. These fat pads were shown to contain ganglion cells histologically. It was observed that vagal effects upon cardiac rate are selectively mediated by neurons located in ganglia overlying the right pulmonary veins at the junction of the right atrium and superior vena cava. On the other hand, vagal effects upon AV conduction were selectively mediated by neurons located in a fat pad at the junction of the inferior vena cava and the inferior left atrium. These pharmacological data support the concept that specific intracardiac ganglia are capable of selective control of SA rate and AV conduction.

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic ganglia on the surface of the heart which are capable of selective control of sino-atrial rate, atrio-ventricular conduction, and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates heart rate (the SA ganglion). Medullary tissues were processed for the histochemical visualization of retrogradely labeled neurons and for the immunohistochemical detection of the neurotransmitter substance P (SP) by dual labeling light and electron microscopic methods. Negative chronotropic retrogradely labeled cells were found in a long slender column in the ventrolateral nucleus ambiguous (NA-VL) which enlarged somewhat at the level of the area postrema. These cells were found bilaterally, but they were asymmetrically distributed. Half the animals showed a pronounced right side predominance in retrograde labeling, while the other half of the animals showed a lesser left side predominance. These observations may help to explain some of the controversy in the literature concerning the relative influence of the right and left vagus nerves on sinus rate. Ultrastructural examination demonstrated axo-somatic and axo-dendritic contacts between SP nerve terminals and retrogradely labeled negative chronotropic NA-VL neurons. SP immunoreactivity was often associated with large dense-core vesicles in terminals forming either symmetric or asymmetric synapses. These observations provide a potential anatomical substrate for the centrally mediated bradycardia elicited by microinjections of SP into the NA. SP immunoreactive terminals were also observed to make axo-somatic, axo-dendritic, and axo-axonic synapses with unlabeled neurons in NA-VL.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide Y-like immunoreactive (NPY-LI) perikarya and processes have been identified in the medulla of the cat. NPY-LI perikarya were found in 4 regions; (1) the medial N. of the solitary tract (MNTS), (2) the lateral tegmental field (LTF), (3) the ventrolateral medullary surface (VLMS), and (4) in the spinal trigeminal nucleus. Tyrosine hydroxylase-like immunoreactive (TH-LI) neurons were also found in the first 3 regions noted above. NPY-LI and TH-LI neurons had a similar morphology and distribution. NPY-LI and TH-LI neurons were counted in sections from 3 representative levels of the medulla. NPY-LI cells were most numerous in the LTF, especially at the level of the area postrema and more rostrally. The lowest number of NPY-LI cells was seen in the MNTS, particularly at a level caudal to the area postrema. NPY-LI cells were at least 3-fold less numerous than TH-LI cells at all levels of the MNTS, but there was a 27-fold greater number of TH-LI neurons at a level of MNTS caudal to the area postrema. In the LTF, NPY-LI cells were 2- to 5-fold less common than TH-LI cells. Approximately equal numbers of NPY-LI and TH-LI cells were counted in the VLMS at the level of the area postrema and rostrally. These data indicate that the majority of TH-LI cells in the MNTS of the cat probably do not contain NPY as a co-transmitter.(ABSTRACT TRUNCATED AT 250 WORDS)

Norepinephrine (NE) concentrations were measured by radioenzymatic assay in microdissected individual laminae of each segment of the cat spinal cord. Norepinephrine was detected in all areas of the spinal gray matter and showed more than a 7-fold difference in concentration between the laminae with the highest and lowest NE. The cervical, thoracic, and lumbosacral spinal regions showed significant interlaminar differences in NE. Intersegmental changes in NE were seen within single laminae of the thoracic and lumbosacral spinal cord, but not in the cervical spinal cord. A significant rostral to caudal, increasing regional gradient of NE was observed from the cervical to lumbosacral spinal cord in laminae I-III, V, VI, VII, and IX. In the intermediolateral cell column (IML), epinephrine concentrations were 2 to 5% of NE. Neither neurotransmitter showed a significant intersegmental variation in the IML. These data should prove useful in further defining the precise role of NE in specific regions of the spinal cord that mediate sensory, motor, autonomic, or propriospinal functions.

Two concentrations of 1-butanol (3000 and 6000 ppm) were administered by inhalation to separate groups of 15 pregnant Sprague-Dawley rats for 7 hr per day throughout gestation; 18 male rats were similarly exposed for 7 hr per day for 6 weeks, and mated to unexposed females. Litters were culled to 4 female and 4 male pups and fostered to untreated controls. From days 10-90, offspring were tested as follows: a) ascent on a wire mesh screen, b) rotorod, c) open field and photoelectrically-monitored activity, d) running wheel, e) avoidance conditioning, and f) operant conditioning. Additionally, brains from 10 offspring at 21 days of age were dissected into cerebrum, cerebellum, brainstem, and midbrain. Each sample was assayed for protein and the neurotransmitters acetylcholine, dopamine, norepinephrine, serotonin, met-enkephalin, beta-endorphin, and substance P. Overall, there were few behavioral or neurochemical alterations detected in the offspring following maternal or paternal exposure to either 3000 or 6000 ppm 1-butanol. This scarcity of effects is important to risk assessment extrapolations drawn from ethanol. Based on the structural similarity of 1-butanol to ethanol and long-standing observations that toxicity to adult animals generally increases with chain length among the alcohols, significant behavioral and neurochemical deviations were predicted. The scarcity of effects from butanol needs to be accounted for in hypotheses relating toxicity to alcohol chain length and in risk assessment extrapolations from findings with ethanol.

The distribution of alpha-melantropin-like material (alpha-MSH) was demonstrated in rat brain using a microdissection technique combined with radioimmunoassay. Highest concentrations of alpha-MSH were noted in the hypothalamus and preoptic area. Particularly high concentrations were observed in the arcuate nucleus, median eminence, suprachiasmatic nucleus, periventricular nucleus and the medial preoptic nucleus. The thalamic paraventricular nucleus also contained high alpha-MSH concentrations. Moderate to low concentrations were noted in other thalamic, septal, amygdaloid and midbrain nuclei. Low concentrations were observed in cortical and striatal regions.

The ultrastructure, afferent input, and sites of termination of neurons containing neuropeptide Y-like immunoreactivity (NPY-LI) were examined in the adult rat nucleus accumbens by using the peroxidase-antiperoxidase (PAP) method. The NPY-LI was seen in sparsely distributed, spindle-shaped perikarya having cross-sectional diameters of 15-20 microns. These perikarya exhibited highly invaginated nuclear membranes and thin rims of cytoplasm containing Golgi lamellae, dense-core vesicles, and other organelles. A few large, principally aspiny, dendrites also showed NPY-LI. The dendrites received synaptic input from unlabeled terminals forming both symmetric and asymmetric junctions. Immunolabeling for NPY was evident in other processes that were not clearly differentiated as dendrites or axons. These were seen primarily near glial processes and the basal laminae of blood vessels. A few myelinated and many unmyelinated axons and axon terminals also were labeled for NPY. These terminals contained numerous, small (40-60 nm), clear and one or more large (80-100 nm) dense core vesicles. Forty-seven percent (27 out of 57) of the terminals containing NPY-LI formed symmetric junctions with unlabeled dendrites or dendritic spines. The remainder lacked recognizable densities within single planes of section. The neurons exhibiting NPY-LI in the nucleus accumbens were characterized further with respect to their afferent input from terminals labeled for the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD). Immunogold labeling of a rabbit antiserum against NPY and PAP labeling for a sheep antiserum to GAD were sequentially applied to the same sections. The GAD-labeled terminals formed symmetric junctions primarily with the more numerous unlabeled dendrites. However, a few synaptic junctions also were detected between the GAD-labeled terminals and dendrites showing immunogold labeling for NPY. We conclude (1) that in the rat nucleus accumbens, NPY-LI is found principally in neurons of the aspiny type and (2) that the output from these presumably intrinsic neurons to other neighboring neurons or blood vessels is at least partially modulated by GABA.

The present study was carried out to determine whether dorsal raphe serotonergic neurons are involved in the regulation of suckling-induced PRL release. Neurotoxin lesions were placed stereotaxically in lactating rats on day 1 of lactation by 5,7-dihydroxytryptamine microinjection into the dorsal raphe (DR), median raphe (MR), or superior colliculus (SC), an area devoid of serotonergic perikarya. Litters were adjusted to eight pups each and weighed daily to determine litter growth rates. On day 7 of lactation, litters were separated from mothers for 8 h, after which six healthy foster pups were provided for a 30-min suckling stimulus. Animals were killed by decapitation immediately after suckling, plasma was collected for RIA of PRL, and brains were frozen and dissected for determination of hypothalamic, caudate, and hippocampal serotonin (5-HT) using the enzymatic-isotopic assay procedure. Litter growth rates from days 1-7 of lactation were significantly different among lesion groups (P less than 0.005), with litters from SC-lesioned animals (SCL) growing similarly to the sham group (sham, 0.924; SCL, 0.941 g/pup . day). In contrast, growth rates of litters from both DR-lesioned (DRL) and MR-lesioned (MRL) animals were significantly depressed (DRL, 0.596; MRL, 0.449 g/pup . day; P less than 0.05 and P less than 0.01, respectively). 5-HT levels in hypothalamus, caudate nuclei, and hippocampus were similar in the sham and SCL groups, whereas hypothalamic 5-HT was depleted by 63% and 55%, respectively, in the DRL and MRL groups. Despite impairments in growth rate and litter survival in both the DRL and MRL groups, only DRL animals showed significant decrements in suckling-induced PRL release (DRL, 288 +/- 107; sham, 837 +/- 134 ng NIAMDD rat PRL RP-1/ml; P less than 0.05) after 5-HT-depleting lesions. The results suggest a specificity of function within the raphe system during lactation; DR 5-HT neurons which project to the hypothalamus provide stimulatory inputs to suckling-induced PRL release, whereas MR 5-HT neurons influence litter growth and survival via their role in maternal behavior.

Binding characteristics of 3H-Substance P (SP) were studied with rat brain membranes using a method applied to peripheral tissues by Lee and Snyder [15]. This method was well applicable to central nervous system (CNS) tissues. The results in the present study indicate that specific 3H-SP binding reaches a plateau only after 20 minutes of incubation, and the binding sites are saturable at a relatively low concentration of 3H-SP. Scatchard analysis of specific binding data reveals a single class of binding sites with a high affinity (Kd = 0.30 nM) and a low density (Bmax = 27.7 fmol/mg protein) in rat brain membranes. A Hill plot of the displacement curve of 3H-SP with unlabelled SP showed no indication for cooperativity (nH = 0.83). The relative potencies of binding of various SP fragments at 3H-SP binding sites were fairly parallel to the length of the C-terminal fragments. Neurotransmitters not structurally related to SP produced no effect on 3H-SP binding even when used at micromolar concentrations.

An extensive system of neuropeptide Y (NPY) containing neurons has recently been identified in the central and peripheral nervous system. In addition, NPY and a structurally related peptide, peptide YY (PYY), containing endocrine cells have been identified in the periphery. The NPY system is of particular interest as the peptide coexists with catecholamines in the central and sympathetic nervous system and adrenal medulla. Evidence has been presented which indicates that NPY may play important roles in regulating autonomic function.

The industrial solvent 2-methoxyethanol (2ME) has antifertility effects in male rats at 300 ppm and is teratogenic in rats and rabbits at 50 ppm. The present research investigated if exposure of paternal or maternal animals to 25 ppm 2ME, the current U.S. permissible occupational exposure limit, would produce detectable effects in the offspring. Eighteen male young-adult Sprague-Dawley rats were exposed to 25 ppm 2ME 7 hr/day, 7 days/week for 6 weeks; they were then mated with untreated females which were allowed to deliver and rear their young. In addition, groups of 15 pregnant rats were exposed 7 hr/day on gestation days 7-13 or 14-20 and allowed to deliver and rear their young. At birth, litters were culled to 4 females and 4 males for behavioral testing of neuromotor function, activity, and simple learning ability on days 10 through 90. In addition, brains from newborn and 21-day-old offspring were analyzed for neurochemical deviations from controls. No effects on paternal or maternal animals, nor on the number or weight of live offspring, were noted. Behavioral testing revealed significant differences from controls only in avoidance conditioning of offspring of mothers exposed on days 7-13. In contrast, neurochemical deviations were observed in brains from 21-day-old offspring from the paternally exposed group as well as from both maternally exposed groups; changes were numerous in the brainstem and cerebrum but were fewer in the cerebellum and midbrain. Thus it appears that both paternal and maternal inhalation of 25 ppm 2ME produces some effect which is reflected in neurochemical deviations in the offspring.

In addition to its widespread social use, ethanol is used extensively as an industrial solvent. Inhalation exposures to ethanol which produce narcosis in maternal rats are not teratogenic. The present study sought to extend the previous research by including offspring from paternal exposures, and testing for behavioral disorders in the offspring following maternal or paternal exposures. Groups of 18 male (approximately 450 g) and 15 female (200-300 g) Sprague-Dawley rats were exposed 7 hours/day for six weeks or throughout gestation to 16000, 10000, or 0 ppm ethanol by inhalation and then mated with untreated rats. Litters were culled to 4 males and 4 females, and were fostered within 16 hours after birth to untreated dams which had delivered their litters within 48 hours previously. Offspring from paternally or maternally exposed animals performed as well as controls on days 10-90 in tests of neuromotor coordination (ascent on a wire mesh screen, rotorod), activity levels (open field, modified-automated open field, and running wheel), and learning ability (avoidance conditioning and operant conditioning). In addition, brains of 10 21-day-old pups were analyzed for neurochemical differences from controls in concentrations of protein and the neurotransmitters acetylcholine, dopamine, norepinephrine, 5-hydroxytryptamine, substance P, Met-enkephalin, and beta-endorphin. Levels of acetylcholine, dopamine, substance P, and beta-endorphin were essentially unchanged in the offspring of rats exposed to ethanol. Complex, but significant changes in levels of norepinephrine occurred only in paternally exposed offspring. 5-Hydroxytryptamine levels were reduced in the cerebrum, and Met-enkephalin levels were increased in all brain regions of offspring from both maternally and paternally exposed rats.

Pharmacological and morphological evidence suggests that the functional activity of serotonergic neurons may be regulated by catecholamines. We have attempted to reveal a potential pathway by which this interaction might occur. Rats received bilateral knife cut lesions of the ventral noradrenergic bundle which severed the A-1 and A-2 cell body contributions to this projection. Controls received a sham lesion into the cerebellum. Two weeks later all animals were sacrificed, and norepinephrine and serotonin levels were measured in discrete nuclei of the brain. Lesion placement was confirmed histofluorometrically. Serotonin levels in the median raphe nucleus were significantly reduced by 40%, but levels of serotonin were unaffected in the dorsal raphe nucleus and 8 serotonergic terminal regions. The lesions did not affect levels of norepinephrine in the locus coeruleus, cingulate cortex, or habenula. This study suggests that a noradrenergic projection to the median raphe nucleus from the A-1 and A-2 cell body groups may modulate serotonergic neuronal function.

Norepinephrine (NE) and dopamine (DA) levels and turnover were measured in 17 discrete brain regions of Swiss-Webster (NIH) mice made aggressive by prolonged isolation. The NE steady state level was significantly lower in olfactory tubercle and substantia nigra and significantly higher in the septal area of the aggressive mice when compared to the isolated non-fighter controls. NE turnover was only higher in the A-10 region of the aggressors. DA steady state level and turnover was lower in olfactory tubercle and higher in caudate putamen of the aggressors. The significance of these changes in isolation-induced aggression is discussed.

In contrast to the pronounced fall in 5HT levels and synaptosomal uptake caused by p-chloroamphetamine, bilateral lesions of the B-9 cell group caused minimal regional changes, except for 35% decreases in the metathalamus-thalamus. We conclude therefore that the prolonged biochemical effects of p-chloroamphetamine are not due to a selective cytotoxic action on B-9 cells; and that a lateral 5HT pathway, possibly from the B-9 cell group, projects to the metathalamus-thalamus.