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We wanted to ascertain whether the lateral parabrachial nucleus was involved in mediating the heart-rate response evoked during stimulation of somatic nociceptors. Reversible inactivation of the lateral parabrachial nucleus, using a GABAAagonist, reduced the reflex tachycardia evoked during noxious (mechanical) stimulation of the forelimb by ∼50%. The same effect was observed after blockade of neurokinin 1 receptors within the lateral parabrachial nucleus, indicating a possible involvement for substance P as a neurotransmitter. Immunocytochemistry revealed a strong expression of substance P-immunoreactive fibers and boutons in all lateral subnuclei, but they were particularly dense in the lateral crescent subnucleus. Histological verification showed that the most effective injection sites for attenuating the noxious-evoked tachycardia were all placed in or near to the lateral crescent nucleus of the lateral parabrachial complex. Many single units recorded from this region were activated by high-intensity brachial nerve stimulation. The brachial nerve evoked firing responses of some of these neurons was reversibly reduced after local delivery of a neurokinin 1 receptor antagonist. However, only a minority of these neurons followed a paired-pulse stimulation protocol applied to the spinal cord, suggesting a predominance of indirect projections from the spinal cord to the parabrachial nucleus. We conclude that the cardiac component of the response to somatic nociception involves indirect spinal pathways that most likely excite neurons located in the lateral crescent nucleus of the parabrachial complex via activation of neurokinin 1 receptors.

In the present study, we investigated in anesthetized rats the influences of the pontine rapid-eye-movement (REM) sleep center on trigeminally induced respiratory responses. We evoked the nasotrigeminal reflex by electrical stimulation of the ethmoidal nerve (EN5) and analyzed the EN5-evoked respiratory suppression before and after injections into the pontine reticular nuclei of the cholinergic agonist carbachol. After injections of 80–100 nl of carbachol (20 mM), we observed a decrease in respiratory rate, respiratory minute volume, and blood pressure but an increase in tidal volume. In those cases in which carbachol injections alone caused these REM sleep-like autonomic responses, we also observed that the EN5-evoked respiratory suppression was significantly potentiated. Unfortunately, carbachol injections failed to depress genioglossus electromyogram (EMG) effectively, because the EMG activity was already strongly depressed by the anesthetic α-chloralose. We assume that pontine carbachol injections in our anesthetized rats cause autonomic effects that largely resemble REM sleep-like respiratory and vascular responses. We therefore conclude that the observed potentiation of EN5-evoked respiratory suppression after carbachol might be due to REM sleep-associated neuronal mechanisms. We speculate that activation of sensory trigeminal afferents during REM sleep might contribute to pathological REM sleep-associated respiratory failures.

In the present study, we investigated the key molecules that determine gamma-aminobutyric acid (GABA)ergic signal transduction in the parabrachial/Kolliker-Fuse complex (PB/KF) by means of immunocytochemistry and in situ hybridization. Our data demonstrate a dense plexus of GABA-immunoreactive (-ir) varicosities throughout the nuclei of the PB and the KF. The number of neurons expressing GAD65 or GAD67 mRNA was fairly low in the PB, whereas caudally in the KF an accumulation of GAD-expressing neurons was observed. The GABA transporter-3 (GAT-3) was detected in all parts of the PB/KF, whereas immunolabeling for GAT1 was not observed. All nuclei of the PB and the KF exhibited immunoreactivity for the gamma2-, alpha2-, and alpha3-subunits of the GABA(A) receptor. Gamma2-ir was strong and similar in all PB/KF nuclei. In contrast, alpha2-labeling was particularly intense in the superior lateral PB, and alpha3-labeling was most prominent in the external lateral and external medial PB, compared with the remaining nuclei. With respect to the subcellular localization, we found gamma2-ir in cell bodies and higher order dendrites, whereas alpha2- and alpha3-ir was predominantly found in cell bodies. Immunolabeling for the beta2/3- and the alpha1-subunit was seen in cell bodies and presumed dendritic profiles. The staining intensity was strongest in the dorsal lateral PB. Most importantly, the external lateral PB and the waist area were totally devoid of beta2/3- and alpha1-ir. Our data suggest that neural processing in the PB/KF is under a strong GABAergic inhibition that is apparently mediated by different types of GABA(A) receptors in functionally different pathways through the PB/KF.

The present study examined the location of neurons in the lateral parabrachial nucleus (PBL), Kölliker-Fuse nucleus (KF), nucleus of the solitary tract (NTS), spinal trigeminal nucleus (Sp5C) and upper cervical cord possibly involved in the transmission of autonomic responses (apnea, bradycardia and rise in arterial blood pressure) elicited by nasotrigeminal stimulation in the rat. To identify these neurons we employed immunocytochemical detection of the transcription factor Fos. To induce the expression of Fos protein, two kinds of stimuli and experimental controls were performed in chloralose/urethane-anesthetized animals: (i) electrical stimulation of the trigeminal ethmoidal nerve (EN5) and, as sham controls, dissection of the EN5 without electrical stimulation, (ii) stimulation of the nasal mucosa with water and, as control experiments, no stimulation. Both forms of stimulation lead to a consistent pattern of Fos-positive neurons in the PBL and KF. Differences could be observed rostrally in the PBL and KF, where significantly higher numbers of Fos-positive neurons were present after EN5 versus water stimulation. The EN5-stimulated group had a significantly higher number of Fos-immunoreactive neurons in the KF than the sham controls, especially in the midlevel region. In the PBL significant differences in the numbers of activated cells could be observed between EN5-stimulated versus sham controls. In the water-stimulated rats compared with the anesthesia controls, a significantly higher number of Fos-immunoreactive neurons was always observed at all rostrocaudal levels in the KF and in the midlevel PBL. Electrical EN5 stimulation induced Fos expression in the Sp5C at the level of the area postrema and caudally in the upper cervical cord. In contrast, after water stimulation Fos-positive neurons were exclusively found in the Sp5C. In addition, all forms of stimuli and controls induced strong expression of Fos in the medial and commissural NTS. Linear correlations were found between the numbers of Fos-immunoreactive neurons in the Sp5C versus the KF and the NTS versus the PBL. The activated neurons may belong to two functionally discrete pathways: the nasotrigeminal reflex circuit, which is activated by nasal sensory afferents running through the EN5 via the Sp5C to the KF, and a pathway activated most likely by baro- and chemoreceptor afferents running through the NTS to the PBL. Our results indicate that the PB/KF plays a pivotal role in the mediation and maintenance of the autonomic responses induced by the nasotrigeminal reflex.

The segmental and laminar organization of spinal projections to the functionally distinct ventrolateral (vlPAG) and lateral periaqueductal gray (lPAG) columns was examined by using retrograde and anterograde tracing techniques. It was found 1) that spinal input to both vlPAG and lPAG columns arose predominantly from neurons in the upper cervical (C1-4) and sacral spinal cord; 2) that there was a topographical separation of vl-PAG projecting and lPAG-projecting neurons within the upper cervical spinal cord; but 3) that below spinal segment C4, vlPAG-projecting and lPAG-projecting spinal neurons were similarly distributed, predominantly within contralateral lamina I, the nucleus of the dorsolateral fasciculus (the lateral spinal nucleus) and the lateral (reticular) part of lamina V. Consistent with the retrograde results, the greatest density of anterograde label, within both the vlPAG and lPAG, was found after tracer injections made either in the superficial or deep dorsal horn of the upper cervical spinal cord. Tracer injections made within the thoraco-lumbar spinal cord revealed that the vlPAG column received a convergent input from both the superficial and deep dorsal horn. However, thoraco-lumbar input to the lPAG was found to arise uniquely from the superficial dorsal horn; whereas the deep dorsal horn was found to innervate the "juxta-aqueductal" PAG region rather than projecting to the lPAG. These findings suggest that similar to spino-parabrachial projections, spinal projections to the lPAG (and juxta-aqueductal PAG) are topographically organised, with distinct subgroups of spinal neurons projecting to specific lPAG or juxta-aqueductal PAG subregions. In contrast, the vlPAG receives a convergent spinal input which arises from the superficial and deep dorsal horn of cervical, thoracic, lumbar, and sacral spinal segments.

THE present study examined whether the Kolliker-Fuse nucleus (KF) plays a role in mediating the trigeminally induced apnoea which occurs after noxious perturbation of the nasal mucosa. We stimulated the ethmoidal nerve (EN5) electrically and recorded respiratory responses before and after injections of the calcium channel blocker CoCl 2 into the KF. Unilateral EN5 stimulations resulted in an apnoea or in a reduction of respiratory frequency and tidal volume. EN5 stimulations immediately after ipsilateral CoCl 2 injections into the caudal KF caused only minor respiratory suppression, indicating a blockade of synaptic transmission. Recovery of the respiratory responses was observed 15-120 min after the CoCl 2 injection. Our data strongly suggest that the caudal KF is an obligatory relay site for trigeminally induced apnoea.

We examined the organization of somatosensory projections to the parabrachial (PB) and Kölliker-Fuse (KF) nuclei by employing the retrograde and anterograde axonal transport of Fluorogold and Phaseolus vulgaris-leucoagglutinin (PHA-L), respectively. Small PHA-L injections were made into different parts of the spinal trigeminal complex, including the paratrigeminal nucleus, and into different segments and laminae of the spinal dorsal horn. The subnuclear distribution of axonal labeling in the PB and KF was mapped with a camera lucida. Our results show that the somatosensory input to the PB and KF is highly organized. Neurons in the spinal trigeminal nuclei project predominantly to the KF and to the ventral portion of the external lateral PB. Neurons in the paratrigeminal nucleus project to the ventral lateral PB, the external medial PB, and to caudal aspects of the medial PB. These findings were supported by retrograde tracing experiments with Fluorogold. Spinal cord neurons located in the superficial dorsal horn (laminae I-II) of upper cervical segments project specifically to the ventral portion of the external lateral PB and, although more sparsely, to various other lateral PB nuclei. In contrast, neurons in the superficial dorsal horn of thoracic and lumbar spinal segments project mainly to the dorsal lateral and the central lateral PB. Finally, neurons in the lateral reticulated area and the lateral spinal nucleus of all spinal segments project almost exclusively to the internal lateral PB, whereas neurons in the respective nuclei of upper cervical segments also project to the KF. From our data we conclude that the somatosensory projections to the PB and KF are topographically organized. It is assumed that these pathways, which run from trigeminal and spinal neurons through the PB and KF to various forebrain, medullary, and spinal nuclei, form functionally different neural circuits that are involved in somatoautonomic processing.

In this study we investigated the effects of possible modulatory transmitters on acoustically responsive neurons of the caudal pontine reticular nucleus (PnC). From previous work in our laboratory it has been suggested that the acoustically responsive giant neurons of this nucleus are the sensorimotor interface mediating the acoustic startle response. Furthermore they are the site of some of the modulatory influence impinging on this response. Besides a possibly glutamatergic excitation from the amygdala a cholinergic input from the midbrain has been described which may use substance P as cotransmitter. Therefore we used electrophysiological and histochemical methods to study this possible modulatory influence in the caudal pontine reticular nucleus. In the first part of this study we recorded extracellularly from single units in the PnC in vivo and studied the effects of iontophoretically applied transmitters. Substance P elicited a long lasting excitation. This excitatory effect of SP was potentiated by acetyl-beta-methylcholine (AMCh, an acetylcholine agonist), whereas single application of AMCh showed no uniform response. Glutamate elicited a potent brief excitation, while application of GABA showed a potent brief inhibition of PnC neurons. In the second part of this study we employed immunoperoxidase staining for substance P, which revealed a fairly dense network of substance P-immunoreactive (SP-ir) fibers in the lateral and ventral aspects of the PnC. Combining retrograde tracing and immunocytochemistry for substance P, we demonstrated that the SP-ir axons in the PnC originate mainly in the laterodorsal tegmental nucleus. We therefore conclude that activation of the laterodorsal tegmental nucleus may facilitate the acoustic startle response by a long lasting excitation of neurons in the caudal pontine reticular nucleus.

The anterograde tracer Phaseolus vulgaris-leucoagglutinin was injected into the medial nucleus of the solitary tract and into the rostral dorsomedial medulla. A sequential two-color immunoperoxidase staining was accomplished in order to demonstrate the co-distribution of presumed terminal axons with chemically distinct neurons in the dorsal raphe nucleus of the midbrain central gray, i.e., B7 serotonergic and A10dc dopaminergic neurons. Black-stained efferent fibers from the medial nucleus of the solitary tract and the rostral dorsomedial medulla intermingled with brown-stained serotonergic (5-hydroxytryptamine-immunoreactive) or dopaminergic (tyrosine hydroxylase-immunoreactive) neurons. Light microscopy revealed that the black-stained efferent axons exhibited numerous en passant and terminal varicosities that were often found in close apposition to brown-stained serotonergic and dopaminergic somata, and to proximal and distal dendrites and dendritic processes. The close association of immunoreactive elements suggests the presence of axo-somatic and axo-dendritic synaptic contacts of medullary fibers with serotonergic and dopaminergic neurons in the dorsal raphe nucleus. These projections could be involved in the modulation of dorsal raphe neurons, depending on the autonomic status of an animal.

The periaqueductal or midbrain central gray matter (CG) in the rat contains a dense network of adrenergic and noradrenergic fibers. We examined the origin of this innervation by using retrograde and anterograde axonal tracers combined with immunohistochemistry for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT). Following injections of the fluorescent tracers Fast Blue or Fluorogold into the CG, double-labeled neurons in the medulla were identified mainly in the noradrenergic A1 group in the caudal ventrolateral medulla (VLM) and A2 group in the medial part of the nucleus of the solitary tract (NTS); and in the adrenergic C1 group in the rostral ventrolateral medulla and C3 group in the rostral dorsomedial medulla. Injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into these cell groups resulted in a distinct pattern of axonal labeling in various subdivisions of the CG. Anterogradely labeled fibers originating in the medial NTS were predominantly found in the lateral portion of the dorsal raphe nucleus and in the adjacent part of the lateroventral CG (CGlv). Following PHA-L injections into the C3 region the anterogradely labeled fibers were diffusely distributed in the CGlv and the dorsal raphe nucleus at caudal levels, but rostrally tended to be located laterally in the CGlv. In contrast, ascending fibers from the caudal and rostral VLM terminated in the rostral dorsal part of the CGlv and in the dorsal nucleus of the CG, whereas ventral parts of the CG, including the dorsal raphe nucleus, contained few afferent fibers. Double-label studies with antisera against DBH and PNMT confirmed that noradrenergic neurons in the A1 and A2 groups and adrenergic neurons in the C1 and C3 groups contributed to these innervation patterns in the CGlv. Noradrenergic and adrenergic projections from the medulla to the CG may play an important role in a variety of autonomic, sensory and behavioral processes.

We investigated projections from the cochlear nucleus in the rat using the anterograde tracer Phaseolus vulgaris-leucoagglutinin. We focused on nuclei in the brainstem which are not considered to be part of the classical auditory pathway. In addition to labeling in auditory nuclei, we found presumed terminal fibers in 4 pontine and mesencephalic areas: (1) the pontine nucleus (PN), which receives bilateral projections from the antero- and posteroventral cochlear nuclei; (2) the ventrolateral tegmental nucleus (VLTg), which receives a contralateral projection from the rostral portion of the anteroventral cochlear nucleus; (3) the caudal pontine reticular nucleus (PnC), which receives bilateral input originating predominantly in the dorsal cochlear nucleus; and (4) the lateral paragigantocellular nucleus (LPGi), which receives projections from all subdivisions of the cochlear nuclei. In the VLTg and PnC, anterogradely labeled varicose axons were often found in close apposition to the primary dendrites and somata of large reticular neurons. Injections of the retrograde fluorescent tracer Fluoro-Gold into the VLTg demonstrated that the neurons of origin are mainly located contralaterally in the rostral anteroventral cochlear nucleus and in the cochlear root nucleus. The relevance of these auditory projections for short-latency audio-motor behaviors and acoustically elicited autonomic responses is discussed.

We examined the monoaminergic innervation of the rat cochlear nucleus (CN) and the inferior colliculus (IC) by using retrograde transport of the fluorescent dye Fluoro-Gold combined with immunohistochemistry. We used antisera against the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), and one against the transmitter serotonin (5-HT). Each substance revealed a distinct pattern of immunoreactive staining in the CN and the IC. In the CN, DBH-immunoreactive (-ir) fibers were present in all subnuclei. The molecular layer of the dorsal CN and the granular layer of the ventral CN, however, were largely devoid of DBH-ir fibers. In contrast, 5-HT-ir fibers were abundant in the molecular layer and the granular cell layer of the CN. In the dorsal CN and the postero- and anteroventral CN, however, this innervation was less dense and evenly distributed across subnuclei. In the IC, the DBH-ir fibers were slightly more numerous in layer 2 of the dorsal cortex than in other subnuclei, while the layer 1 of both the dorsal and the external cortex contained only a few fibers. In contrast, the 5-HT-ir fibers formed a dense network in both the dorsal and external cortices of the IC, while they were less abundant in the remaining subnuclei. PNMT-ir fibers were not found in any of the auditory brainstem nuclei. Following Fluoro-Gold injections into the CN or IC, retrogradely labeled DBH-ir neurons were found in the A6 noradrenergic cell group (locus coeruleus). The CN received additional projections from the A5 noradrenergic cell group, as well as sparse projections from the A4 and A7 cell groups. The serotonergic innervation of the CN and IC originated largely in the B7 serotonergic cell group (dorsal raphe nucleus). Serotonergic neurons in other groups of the raphe nuclei were only occasionally labeled. Our data indicate that both noradrenaline and serotonin may play a role in central auditory processing. Their differential distribution in the IC and CN subnuclei suggests that these transmitter systems might influence different functional circuits.

On the basis of stimulation studies, it has been proposed that the infralimbic cortex (ILC), Brodmann area 25, may serve as an autonomic motor cortex. To explore this hypothesis, we have combined anterograde tracing with Phaseolus vulgaris leucoagglutinin (PHA-L) and retrograde tracing with wheat germ aggutinin conjugated to horseradish peroxidase (WGA-HRP) to determine the efferent projections from the ILC. Axons exit the ILC in one of three efferent pathways. The dorsal pathway ascends through layers III and V to innervate the prelimbic and anterior cingulate cortices. The lateral pathway courses through the nucleus accumbens to innervate the insular cortex, the perirhinal cortex, and parts of the piriform cortex. In addition, some fibers from the lateral pathway enter the corticospinal tract. The ventral pathway is by far the largest and innervates the thalamus (including the paraventricular nucleus of the thalamus, the border zone between the paraventricular and medial dorsal nuclei, and the paratenial, reuniens, ventromedial, parafasicular, and subparafasicular nuclei), the hypothalamus (including the lateral hypothalamic and medial preoptic areas, and the suprachiasmatic, dorsomedial, and supramammillary nuclei), the amygdala (including the central, medial, and basomedial nuclei, and the periamygdaloid cortex) and the bed nucleus of the stria terminalis. The ventral efferent pathway also provides descending projections to autonomic cell groups of the brainstem and spinal cord including the periaqueductal gray matter, the parabrachial nucleus, the nucleus of the solitary tract, the dorsal motor vagal nucleus, the nucleus ambiguus, and the ventrolateral medulla, as well as lamina I and the intermediolateral column of the spinal cord. The ILC has extensive projections to central autonomic nuclei that may subserve a role in modulating visceral responses to emotional stimuli, such as stress.

We examined the organization of descending projections from auditory and adjacent cortical areas to the inferior colliculus (IC) in the rat by using the retrograde and anterograde transport of wheat germ agglutinin-horseradish peroxidase. Small tracer injections were placed into cytologically defined subnuclei of the IC. On the basis of the resulting pattern of retrogradely labeled neurons in the cortex, different cortical areas and fields were defined. Two secondary areas located ventrocaudally (Te2) and ventrally (Te3) to the primary auditory area (Te1) were delineated. The primary auditory area was subdivided into a posterior (Te1.p), a medial (Te1.m), and an anterior (Te1.a) auditory field. In addition, we outlined an area located rostrally to the auditory areas comprising a part of the secondary somatosensory cortex, as well as a dorsal belt surrounding dorsally the auditory areas. The following basic patterns of corticocollicular projections are revealed: (1) layers 2 and 3 of the dorsal cortex of the IC (DC2, DC3) are differentially innervated by the primary auditory fields (Te1.p and Te1.a project bilaterally to DC2, while Te1.m projects bilaterally and in topographical order to DC3); cells in Te1.m, arranged in caudal to rostral sequence, project to corresponding loci in DC3 arranged from dorsolateral to ventromedial; (2) the fibrocellular capsule of the IC, comprising layer 1 of the dorsal and external cortex of the IC, receives input from the secondary auditory area Te2; (3) layers 2 and 3 of the external cortex of the IC are only weakly innervated by the primary and secondary auditory cortex; (4) the intercollicular zone receives its major input from the secondary auditory area Te3, the secondary somatosensory cortex, and the dorsal belt; and (5) finally, the central nucleus of the IC receives no input from the temporal cortex at all. Our results demonstrate that the corticocollicular projections are highly organized. These pathways may modulate auditory processing in different functional circuits of the inferior colliculus.

In a previous study (Herbert et al., J. Comp. Neurol. [1990];293:540-580), we demonstrated that the ascending afferent projections from the medulla to the parabrachial nucleus (PB) mark out functionally specific terminal domains within the PB. In this study, we examine the organization of the forebrain afferents to the PB. The PB was found to receive afferents from the infralimbic, the lateral prefrontal, and the insular cortical areas; the dorsomedial, the ventromedial, the median preoptic, and the paraventricular hypothalamic nuclei; the dorsal, the retrochiasmatic, and the lateral hypothalamic areas; the central nucleus of the amygdala; the substantia innominata; and the bed nucleus of the stria terminalis. In general, forebrain areas tend to innervate the same PB subnuclei from which they receive their input. Three major patterns of afferent termination were noted in the PB; these corresponded to the three primary sources of forebrain input to the PB: the cerebral cortex, the hypothalamus, and the basal forebrain. Hypothalamic afferents innervate predominantly rostral portions of the PB, particularly the central lateral and dorsal lateral subnuclei. The basal forebrain projection to the PB ends densely in the external lateral and waist subnuclei. Cortical afferents terminate most heavily in the caudal half of the PB, particularly in the ventral lateral and medial subnuclei. In addition, considerable topography organization was found within the individual projections. For example, tuberal lateral hypothalamic neurons project heavily to the central lateral subnucleus and lightly to the waist area; in contrast, caudal lateral hypothalamic neurons send a moderately heavy projection to both the central lateral and waist subnuclei. Our results show that the forebrain afferents of the PB are topographically organized. These topographical differences may provide a substrate for the diversity of visceral functions associated with the PB.

The parabrachial nucleus (PB) is the main relay for ascending visceral afferent information from the nucleus of the solitary tract (NTS) to the forebrain. We examined the chemical organization of solitary-parabrachial afferents by using combined retrograde transport of fluorescent tracers and immunohistochemistry for galanin (GAL), cholecystokinin (CCK), and corticotropin-releasing factor (CRF). Each peptide demonstrated a unique pattern of immunoreactive staining. GAL-like immunoreactive (-ir) fibers were most prominent in the "waist" area, the inner portion of external lateral PB, and the central and dorsal lateral PB subnuclei. Additional GAL-ir innervation was seen in the medial and external medial PB subnuclei. GAL-ir perikarya were observed mainly rostrally in the dorsal lateral, superior lateral, and extreme lateral PB. CCK-ir fibers and terminals were most prominent in the outer portion of the external lateral PB; some weaker labeling was also present in the central lateral PB. CCK-ir cell bodies were almost exclusively confined to the superior lateral PB and the "waist" area, although a few cells were seen in the Kölliker-Fuse nucleus. The distribution of CRF-ir terminal fibers in general resembled that of GAL, but showed considerably less terminal labeling in the lateral parts of the dorsal and central lateral PB, and the external medial and Kölliker-Fuse subnuclei. The CRF-ir cells were most numerous in the dorsal lateral PB and the outer portion of the external lateral PB; rostrally, scattered CRF-ir neurons were seen mainly in the central lateral PB. After injecting the fluorescent tracer Fast Blue into the PB, the distribution of double-labeled neurons in the NTS was mapped. GAL-ir cells were mainly located in the medial NTS subnucleus; 34% of GAL-ir cells were double-labeled ipsilaterally and 7% contralaterally. Conversely, 17% of the retrogradely labeled cells ipsilaterally and 16% contralaterally were GAL-ir. CCK-ir neurons were most numerous in the dorsomedial subnucleus of the NTS and the outer rim of the area postrema. Of the CCK-ir cells, 68% in the ipsilateral and 10% in the contralateral NTS were double-labeled, whereas 15% and 10%, respectively, of retrogradely labeled cells were CCK-ir. In the area postrema, 36% of the CCK-ir cells and 9% of the Fast Blue cells were double-labeled. CRF-ir neurons were more widely distributed in the medial, dorsomedial, and ventrolateral NTS subnuclei, but double-labeled cells were mainly seen in the medial NTS.(ABSTRACT TRUNCATED AT 400 WORDS)

Lesion or pharmacological manipulation of the dorsolateral pons can transform the breathing pattern to apneusis (pathological prolonged inspiration). Apneusis reflects a disturbed inspiratory off-switch mechanism (IOS) leading to a delayed phase transition from inspiration to expiration. Under intact conditions the IOS is irreversibly mediated via activation of postinspiratory (PI) neurons within the respiratory network. In parallel, populations of laryngeal premotoneurons manifest the IOS by a brief glottal constriction during the PI phase. We investigated effects of pontine excitation (glutamate injection) or temporary lesion after injection of a GABA-receptor agonist (isoguvacine) on the strength of PI-pool activity determined from respiratory motor outputs or kinesiological measurements of laryngeal resistance in a perfused brainstem preparation. Glutamate microinjections into distinct parts of the pontine Kolliker-Fuse nucleus (KF) evoked a tonic excitation of PI-motor activity or sustained laryngeal constriction accompanied by prolongation of the expiratory phase. Subsequent isoguvacine microinjections at the same loci abolished PI-motor or laryngeal constrictor activity, triggered apneusis and established a variable and decreased breathing frequency. In summary, we revealed that excitation or inhibition of defined areas within the KF activated and blocked PI activity and, consequently, IOS. Therefore, we conclude, first, that descending KF inputs are essential to gate PI activity required for a proper pattern formation and phase control within the respiratory network, at least during absence of pulmonary stretch receptor activity and, secondly, that the KF contains large numbers of laryngeal PI premotor neurons that might have a key role in the regulation of upper airway resistance during reflex control and vocalization.

Breathing is constantly modulated by afferent sensory inputs in order to adapt to changes in behaviour and environment. The pontine respiratory group, in particular the Kolliker-Fuse nucleus, might be a key structure for adaptive behaviours of the respiratory network. Here, we review the anatomical connectivity of the Kolliker-Fuse nucleus with primary sensory structures and with the medullary respiratory centres and focus on the importance of pontine and medullary postinspiratory neurones in the mediation of respiratory reflexes. Furthermore, we will summarise recent findings from our group regarding ontogenetic changes of respiratory reflexes (e.g., the diving response) and provide evidence that immaturity of the Kolliker-Fuse nucleus might account in neonates for a lack of plasticity in sensory evoked modulations of respiratory activity. We propose that a subpopulation of neurones within the Kolliker-Fuse nucleus represent command neurones for sensory processing which are capable of initiating adaptive behaviour in the respiratory network. Recent data from our laboratory suggest that these command neurones undergo substantial postnatal maturation.

In the present study, we investigated the expression and distribution of key molecules in the parabrachial (PB) and Kölliker-Fuse nuclei (KF) that determine glycinergic signal transduction. By means of immunocytochemistry, we analyzed the amino acid glycine (Gly), the glycine transporters 1 and 2 (GlyT1, GlyT2), and the ligand binding glycine receptor-subunit alpha 1 (GlyR alpha 1). Gly-immunoreactivity (-ir) was mainly found in varicose fibers and presumed terminal boutons; Gly-ir cell bodies were only occasionally seen. Immunoreactivity for GlyT2 was located in axons while GlyT1-staining was diffuse in the neuropil. Immunolabeling for GlyR alpha 1 occurred mostly as granular staining diffusely distributed throughout the neuropil. Only in the superior lateral PB, the lateral crescent of the PB, and caudally in the KF did GlyR alpha 1-ir outline cell bodies and primary and higher-order dendrites. Furthermore, our data demonstrate a distinct codistribution of immunoreactivities for Gly, GlyT2. and GlyR alpha 1 in a specific set of PB nuclei and in the KF. Strong staining was consistently seen in the internal lateral PB, the ventral lateral PB, the lateral crescent, the medial PB adjacent to the superior cerebellar peduncle, and the rostral two-thirds of the KF. Moderate to weak immunostaining was present in the superior, central, and dorsal lateral PB, the external medial PB, the medioventral part of the medial PB, and caudally in the KF. In contrast, remaining nuclei such as the external lateral PB and the waist area were essentially devoid of Gly-ir profiles, GlyT2-ir, and GlyR alpha 1-ir. Immunoreactivity for GlyT1 was evenly distributed throughout all nuclei of the medial and lateral PB, including the external lateral PB and the waist area, while the KF was only weakly stained. Our data provide evidence that glycinergic mechanisms might play a role for neural processing in most nuclei of the PB and in the KF. Only the external lateral PB and the waist area are apparently not subject to glycinergic inhibition.

The aim of the present study was to analyze the occurrence and distribution of flip/flop splice variants of AMPA-type glutamate receptors (GluRA-D) in the rat parabrachial and Kolliker-Fuse nuclei (PB/KF). We performed in situ hybridization experiments on sections through different rostro-caudal levels of the PB/KF and analyzed the subunit expression semiquantitatively by means of grain counts for each probe in eight PB nuclei and in the KF. Our experiments revealed that the splice variants of the AMPA receptor subunit mRNAs are expressed differentially in the distinct nuclei of the PB/KF. The flip splice variants were predominantly expressed (GluRB-D flip) while the flop splice variants (GluRA flop and C flop) were expressed considerably weaker. Within the PB/KF, several nuclei expressed transcripts of GluRB flip (superior, central, dorsal, external, and ventral lateral PB, waist area, medial PB, KF) and GluRC flip (internal, superior, central, dorsal, external, and ventral lateral PB, waist area, KF). GluRB transcripts were not found in neurons of the internal lateral PB and in only 50% of the neurons in the KF. A more restricted expression in the PB/KF was observed for the GluRD flip (internal lateral PB), GluRA flop (medial PB, KF) and GluRC flop mRNA (superior lateral PB, KF). The present data demonstrate that the nuclei of the PB/KF show a differential expression of AMPA receptor subunits. This suggests that the anatomically and functionally distinct nuclei might make use of AMPA-type glutamate receptors with different physiological properties and ion selectivities.

In the present study, we investigated the expression of N-methyl-D-aspartate (NMDA) receptor subunits in the parabrachial/Kolliker-Fuse complex (PB/KF), the intertrigeminal region, the ventrolateral pons, the nucleus of the solitary tract, the ventrolateral medulla with the ambiguus nucleus, and the caudal spinal trigeminal nucleus, which are presumably involved in mediating the autonomic responses to nasotrigeminal stimulation (diving response). Our immunocytochemical data demonstrate that the majority of neurons in the respective nuclei stain for the NR1 subunit, which is a mandatory component of all NMDA receptors. NR1 immunoreactivity was found mainly on neuronal cell bodies and primary dendrites. The ubiquitous expression of the NR1 subunit was confirmed by in situ hybridization, revealing a strong NR1 mRNA signal over neurons in all nuclei investigated. Among the NR2A–D subunits, the strongest expression was observed for the NR2D transcript, both in the PB/KF and in the brainstem. For the PB/KF, we found in addition a moderate expression of NR2A mRNA in the internal lateral PB and of NR2B mRNA in the external lateral PB. The remaining PB nuclei and the KF were essentially devoid of NR2A–C transcripts. For the nucleus of the solitary tract and in the spinal trigeminal nucleus, we found, in addition to the strong NR2D mRNA signal, moderate expression of the NR2A–C transcripts. In the ventrolateral medulla, a moderate signal was seen for NR2C transcript, whereas signals for the NR2A and -B subunits were negligible. Our data suggest that, in PB/KF and pontomedullary brainstem nuclei involved in mediating the diving response, glutamatergic neurotransmission is apparently mediated through a specific type of NMDA receptor channels, consisting essentially of NR1 and NR2D subunits. J. Comp. Neurol. 415:501–517, 1999. © 1999 Wiley-Liss, Inc.

In the present study, we examined the distribution of neurons in the parabrachial nucleus (PB), the Kölliker-Fuse nucleus (KF), the spinal trigeminal nucleus caudalis (Sp5C), the nucleus of the solitary tract (NTS) and the ventrolateral medulla (VLM), which are activated by evoking the nasotrigeminal reflex and which exhibit immunoreactivity for the N-methyl-D-aspartate (NMDA) receptor subunit NR1. By stimulating the nasal mucosa with saline, we induced the expression of the immediate early gene c-fos and combined the immunocytochemical detection of the Fos protein with the detection of the NR1 subunit. Cell counts revealed that nasal stimulation, compared to anesthesia controls, resulted in highly significant increases (por = 0.001) of Fos-immunoreactive (-ir) neurons in the midlevel KF, the external lateral PB, and the Sp5C. In the central lateral PB, the rostral ventrolateral medulla including the Bötzinger/pre-Bötzinger complex, and in the ventrolateral and commissural NTS the increases were only moderately significant (por = 0.05). With respect to the numbers of NR1-/Fos-ir double-labeled neurons, significant increases were only observed in a subset of these pontomedullary nuclei. Increases were highly significant in the Sp5C (por = 0.001) and the midlevel KF (por = 0.01) and moderately significant (por = 0.05) in the external lateral PB, Bötzinger/pre-Bötzinger complex, and ventrolateral NTS. The present study revealed that nasotrigeminally activated neurons in mandatory and potential relay sites of the nasotrigeminal reflex circuit express the NR1 subunit. This finding strongly suggests that NMDA-type glutamate receptors are involved in the mediation of the nasotrigeminally evoked cardiovascular and respiratory responses.

The present study examined the organization of afferent and efferent connections of the rat ventrolateral tegmental area (VLTg) by employing the retrograde and anterograde axonal transport of Fluorogold and Phaseolus vulgaris-leucoagglutinin, respectively. Our interest was focused on whether the anatomical connections of the VLTg would provide evidence as to the involvement of this reticular area in audiomotor behavior. Our retrograde experiments revealed that minor inputs to the VLTg arise in various telencephalic structures, including the cerebral cortex. Stronger projections originate in the lateral preoptic area, the zona incerta, the nucleus of the posterior commissure and some other thalamic areas, the lateral substantia nigra, the deep layers of the superior colliculus, the dorsal and lateral central gray, the deep mesencephalic nucleus, the paralemniscal zone, the intercollicular nucleus, the external cortex of the inferior colliculus, the oral and caudal pontine reticular nucleus, the deep cerebellar nuclei, the gigantocellular and lateral paragigantocellular reticular nuclei, the prepositus hypoglossal nucleus, the spinal trigeminal nuclei, and the intermediate layers of the spinal cord. Most importantly, we disclosed strong auditory afferents arising in the dorsal and ventral cochlear nuclei and in the cochlear root nucleus. The efferent projections of the VLTg were found to be less widespread. Telencephalic structures do not receive any input from the VLTg. Moderate projections were seen to diencephalic reticular areas, the zona incerta, the nucleus of the posterior commissure, and to various other thalamic areas. The major VLTg projections terminate in the deep layers of the superior colliculus, the deep mesencephalic nucleus, the intercollicular nucleus and external cortex of the inferior colliculus, the oral and caudal pontine reticular nucleus, the gigantocellular and lateral paragigantocellular reticular nuclei, and in the medial column of the facial nucleus. From our data, we conclude that the VLTg might play a role in sensorimotor behavior.

Golgi-staining, retrograde and anterograde tract-tracing, and a two-color immunoperoxidase technique have been employed, at the light- and electron-microscopic levels, to analyze the auditory projections from the cochlear nucleus (CN) to the lateral paragigantocellular reticular nucleus (LPGi) in the rat. We have found that the auditory input originates predominantly in the posteroventral and cochlear root nuclei. The auditory axons terminate in the cell-poor, ventral portion of the LPGi, which is strongly invaded by beaded dendritic profiles, originating from parent cell bodies located in the dorsal half of the LPGi. Ultrastructural analysis has revealed that the anterogradely labeled auditory axons form functional synapses preferentially with dendritic shafts. These axo-dendritic contacts are apparently excitatory in nature. By means of a sequential two-color immunoperoxidase staining method, we have further characterized potential postsynaptic neurons in the LPGi. Black-stained auditory fibers intermingle with brown-stained serotonergic or adrenergic neurons. Varicose auditory axons are often closely apposed to immunoreactive dendritic profiles of serotonergic and adrenergic neurons, indicating the presence of possible synaptic contacts of auditory terminal fibers with these transmitter-classified cells. The monosynaptic auditory in- put from the CN may modulate the activity of B3 sero-tonergic and C1 adrenergic cells in the LPGi and may thus induce adaptive changes in response to acoustic stimuli.

The present study describes the distribution of alpha 2-adrenoceptors in the parabrachial and Kölliker-Fuse nucleus of the rat by employing the tritium-labeled alpha 2-receptor antagonist rauwolscine ([3H]-RAUW) as a ligand. The [3H]-RAUW binding was densitometrically quantified in five nuclei of the parabrachial (PB) complex in serial coronal sections. We found that cytoarchitectonically and anatomically distinct nuclei of the PB complex exhibit different numbers of [3H]-RAUW-binding sites. The largest number of binding sites was observed over the external lateral PB and caudally over the waist area of the PB. Lower numbers of binding sites were found in the remaining lateral PB nuclei, followed by the medial PB and the Kölliker-Fuse nucleus. In addition we disclosed that the internal lateral PB contains a very low number of binding sites while the external medial PB is marked by dense [3H]-RAUW binding. Also, the affinities of the binding sites differed between the PB areas. High affinities were observed in the external lateral PB, the remaining lateral PB nuclei and in the waist area of the PB, while the medial PB and the Kölliker-Fuse nucleus exhibited only low affinities for the ligand. Furthermore, saturation curves demonstrated non-linear profiles, indicating the presence of more than one population of binding sites in the PB nuclei for the radioligand. Our data demonstrate that the PB exhibits a distinct distribution of alpha 2-adrenergic binding sites. These correlate well with the cytoarchitectonically defined nuclei of the PB complex and with the pattern of ascending axons from the medial nucleus of the solitary tract and the area postrema terminating in the PB. Since a large number of these projection neurons utilize adrenaline or noradrenaline as their transmitters, we conclude that solitary-parabrachial neurotransmission to the forebrain is, at least in part, mediated via alpha 2-adrenoceptors.

We examined the organization of descending projections from the inferior colliculus (IC) to auditory brainstem nuclei and to pontine and reticular nuclei in the rat by employing the anterograde axonal tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Small PHA-L injections into cytologically defined subnuclei of the IC revealed that each subnucleus has a unique pattern of efferent projections. The central nucleus of the IC projects in a topographic order to the dorsal nucleus of the lateral lemniscus (DLL), the rostral periolivary nucleus (RPO), the ventral nucleus of the trapezoid body (VNTB), and the dorsal cochlear nucleus (DCN). It is assumed that this topography represents a cochleotopic arrangement. The external cortex of the IC projects to the nucleus sagulum (Sag), the RPO, the VNTB, and the DCN. Minor projections were found to pontine and reticular nuclei. Efferent fibers from the dorsal cortex of the IC terminate mainly in the Sag, while other nuclei of the auditory and extra-auditory brainstem receive only minor projections. The intercollicular zone sends a moderate number of fibers to the DLL and very few, if any, to the remaining auditory brainstem nuclei. In contrast, fairly strong projections from the intercollicular zone to the reticular formation were found. The present data demonstrate that the four subnuclei of the IC have a differential pattern of descending projections to nuclei in the pontine and medullary brainstem. These parallel colliculofugal pathways, assumed to belong to functionally separate circuits, may modulate auditory processing at different levels of the auditory neuraxis.

The amplitude of the acoustic startle response (ASR) is markedly reduced when the startle eliciting pulse is preceded by a weak, non-startling stimulus at an appropriate lead time, usually about 100 ms. This phenomenon is termed prepulse inhibition (PPI) and has received considerable attention in recent years as a model of sensorimotor gating. We report here on experiments which were undertaken in order to investigate some of the neural mechanisms of PPI. We focused on the characterization of the cholinergic innervation of the pontine reticular nucleus, caudal part (PnC), an obligatory relay station in the primary startle pathway. The combination of retrograde tracing with choline acetyltransferase-immunocytochemistry revealed a cholinergic projection from the pedunculopontine tegmental nucleus (PPTg) and laterodorsal tegmental nucleus (LDTg) to the PnC. Extracellular recording from single PnC units, combined with microiontophoretic application of the acetylcholine (ACh) agonists acetyl-beta-methylcholine (AMCH) and carbachol revealed that ACh inhibits the majority of acoustically responsive PnC neurons. Neurotoxic lesions of the cholinergic neurons of the PPTg significantly reduced PPI without affecting the ASR amplitude in the absence of prepulses. No effect on long-term habituation of the ASR was observed. The present data indicate that the pathway mediating PPI impinges upon the primary acoustic startle circuit through an inhibitory cholinergic projection from the PPTg to the PnC.

We examined the subnuclear organization of projections to the parabrachial nucleus (PB) from the nucleus of the solitary tract (NTS), area postrema, and medullary reticular formation in the rat by using the anterograde and retrograde transport of wheat germ agglutinin-horseradish peroxidase conjugate and anterograde tracing with Phaseolus vulgaris-leucoagglutinin. Different functional regions of the NTS/area postrema complex and medullary reticular formation were found to innervate largely nonoverlapping zones in the PB. The general visceral part of the NTS, including the medial, parvicellular, intermediate, and commissural NTS subnuclei and the core of the area postrema, projects to restricted terminal zones in the inner portion of the external lateral PB, the central and dorsal lateral PB subnuclei, and the "waist" area. The dorsomedial NTS subnucleus and the rim of the area postrema specifically innervate the outer portion of the external lateral PB subnucleus. In addition, the medial NTS innervates the caudal lateral part of the external medial PB subnucleus. The respiratory part of the NTS, comprising the ventrolateral, intermediate, and caudal commissural subnuclei, is reciprocally connected with the Kolliker-Fuse nucleus, and with the far lateral parts of the dorsal and central lateral PB subnuclei. There is also a patchy projection to the caudal lateral part of the external medial PB subnucleus from the ventrolateral NTS. The rostral, gustatory part of the NTS projects mainly to the caudal medial parts of the PB complex, including the "waist" area, as well as more rostrally to parts of the medial, external medial, ventral, and central lateral PB subnuclei. The connections of different portions of the medullary reticular formation with the PB complex reflect the same patterns of organization, but are reciprocal. The periambiguus region is reciprocally connected with the same PB subnuclei as the ventrolateral NTS; the rostral ventrolateral reticular nucleus with the same PB subnuclei as both the ventrolateral (respiratory) and medial (general visceral) NTS; and the parvicellular reticular area, adjacent to the rostral NTS, with parts of the central and ventral lateral and the medial PB subnuclei that also receive rostral (gustatory) NTS input. In addition, the rostral ventrolateral reticular nucleus and the parvicellular reticular formation have more extensive connections with parts of the rostral PB and the subjacent reticular formation that receive little if any NTS input. The PB contains a series of topographically complex terminal domains reflecting the functional organization of its afferent sources in the NTS and medullary reticular formation.

The echolocation behavior ofRhinolophus rouxi when waiting for insects on a perch and when pursuing them in short hunting flights is described. It reveals that flycatcher-style hunting is one foraging strategy utilized by this species. It also suggests that bats use fluttering target information to classify their prey.

The location and number of motoneurons to individual pinna muscles were determined by retrograde transport of horseradish peroxidase in rat and flying fox. The degree of ear mobility differs considerably between these species in that rats perform simpler ear movements while flying foxes move their pinnae in a sophisticated way. Five pinna muscles were investigated in each species. Motoneurons lay within the medial subdivision of the facial motor nucleus extending over its entire rostrocaudal length. They were topographically organized; however, a somatotopic order could not be observed. With one exception homologous pinna muscles were represented in corresponding areas in both species, supporting the idea of a common representation of ear muscles in mammals. In rat, motoneuron pools overlapped considerably, whereas in flying fox overlap was minute. A total of 1,110 and 1,646 motoneurons were labeled in rat and flying fox, respectively. We conclude that the higher number of pinna motoneurons in the latter species in addition to the more clear-cut topography provide the structural substrates that underlie differences in the quality of ear movements as seen in bats vis-a-vis other mammals.

In mammals, the respiratory rhythm is generated by a brainstem neuronal network which produces a continuous pattern of burst discharges that drive the motoneurons of respiratory muscles (Bianchi et al. 1995). This vital rhythm is strongly modulated by sensory afferents arising from the upper and lower airways and also, underlies behavioural and homeostatic changes. A brain area that profoundly modulates the respiratory rhythm is the pontine Kolliker-Fuse (KF) nucleus, characterized as the pontine pneumotaxic centre (Dick et al. 1994; Fung et al. 1994). This refers to the potent influence of the KF on the duration and termination of respiratory phases involving pulmonary afferents. The KF participates also in processing respiratory reflexes, such as the Hering-Breuer reflex (Feldman et al. 1976; Shaw et al. 1989) the chemoreceptor reflex (Koshiya & Guyenet, 1994), the sneeze reflex (Wallois et al. 1995) and the nasotrigeminal reflex (Dutschmann & Herbert, 1996, 1997). Anatomical findings verified the connections of the KF revealing prominent inputs from distinct regions of the nucleus of the solitary tract (NTS) and the ventrolateral medulla (e.g. Herbert et al. 1990) and from spinal and trigeminal neurons which are the primary relays for sensory afferent information from the upper airways and the face (Panneton et al. 1994; Feil & Herbert, 1995). In the present study, we focus on the role of the KF in mediating the nasotrigeminal reflex. The reflex responses can be evoked by noxious stimulation of the nasal mucosa and comprise apnoea in the expiratory state, activation of laryngeal adductor muscles, bradycardia, and peripheral vasoconstriction, leading to rise in arterial blood pressure (Kratschmer, 1870). Consequently, this vital reflex prevents invasion of noxious substances into the upper airways and, moreover, leads to a reduction in oxygen consumption thereby preventing a rapid progression of asphyxia. The nasotrigeminal reflex plays a key role in the diving response of aquatic mammals which is induced by face immersion (Daly, 1984; Elsner & Daly, 1988). The reflex is mediated by the ethmoidal nerve (EN5), a branch of the ophthalmic division of the trigeminal nerve (Sant'Ambrogio et al. 1995) whose fibres terminate in the pars caudalis of the spinal trigeminal nucleus (Sp5C; Anton & Peppel, 1991; Panneton, 1991). Thus, neurons in the Sp5C represent the first central relay for this reflex circuit (Panneton & Yavari, 1995). Recently, we have provided the first experimental evidence that the KF is also a crucial relay site in the nasotrigeminal reflex circuit, in particular for the trigeminally induced apnoea (Dutschmann & Herbert, 1996). We proposed that the KF might represent the sensory-autonomic interface that relays the trigeminal input from the nasal mucosa to cardiorespiratory neurons in the medulla or spinal cord. Histological studies from our laboratory and from others demonstrated in the KF immunoreactivities for various GABAA, glycine and NMDA receptor subunits (Guthmann et al. 1996; Herbert et al. 1996; Guthmann et al. 1997) and for AMPA receptor subunits (Chamberlin & Saper, 1995). Therefore, we analysed the roles of NMDA, AMPA/kainate, GABAA and glycine receptors in the KF for the mediation of the nasotrigeminal reflex responses. We performed microinjections of receptor-specific antagonists into the KF and compared the autonomic responses with electrical EN5 stimulation before and after drug injection.