Catecholaminergic neurons in synaptic connections with pre-Bötzinger complex neurons in the rostral ventrolateral medulla in normoxic and daily acute intermittent hypoxic rats.

The rostral ventrolateral medulla (RVLM) contains cardiovascular-related catecholaminergic neurons and respiratory-related pre-Bötzinger complex (pre-BötC) neurons, which are intermingled and functionally connected for coordinating cardiorespiratory activities. Daily acute intermittent hypoxia (dAIH) is known to elicit respiratory plasticity. However, it is unclear if the catecholaminergic neurons directly synapse onto pre-BötC neurons, and if the local circuitry exhibits plasticity when exposed to dAIH. The present study was aimed to determine the synaptic phenotypes between dopamine-ß-hydroxylase (DßH)-immunoreactive (ir) catecholaminergic neurons and neurokinin 1 receptor (NK1R)-ir pre-BötC neurons, and the effect of dAIH on the neuronal network. Immunofluorescence histochemistry was used to reveal immunoreactivities of DßH and NK1R in the RVLM of normoxic and dAIH rats. Synaptic phenotypes were examined with double-labeling immunoelectron microscopy. We found that DßH immunoreactivity was expressed in somata and processes, some of which were in close apposition to NK1R-ir pre-BötC neurons. DßH-ir gold particles were localized to somata, dendrites, and axonal terminals. DßH-ir terminals formed asymmetric synapses, and occasionally, symmetric synapses in the pre-BötC, featuring the local circuitry. Of the synapses, 28% in normoxic and 29.6% in dAIH groups were apposed to NK1R-ir dendrites. Significant increases in DßH expression and NK1R-ir processes were found in the dAIH group. Moreover, the area and number of processes in close appositions were significantly elevated, strongly suggesting that dAIH induced plasticity with increased connections and interactions between the cardiovascular- and respiratory-related neurons in the local circuitry. In conclusion, asymmetric synapses are predominant in the crosstalk between catecholaminergic and pre-BötC neurons in the RVLM, elaborating excitatory transmission driving the coupling of cardiorespiratory activities. The neural network manifests plasticity in response to dAIH challenge.

Anatomical and functional pathways of rhythmogenic inspiratory premotor information flow originating in the pre-Bötzinger complex in the rat medulla.

The pre-Bötzinger complex (preBötC) of the ventrolateral medulla is the kernel for inspiratory rhythm generation. However, it is not fully understood how inspiratory neural activity is generated in the preBötC and propagates to other medullary regions. We analyzed the detailed anatomical connectivity to and from the preBötC and functional aspects of the inspiratory information propagation from the preBötC on the transverse plane of the medulla oblongata. Tract-tracing with immunohistochemistry in young adult rats demonstrated that neurokinin-1 receptor- and somatostatin-immunoreactive neurons in the preBötC, which could be involved in respiratory rhythmogenesis, are embedded in the plexus of axons originating in the contralateral preBötC. By voltage-imaging in rhythmically active slices of neonatal rats, we analyzed origination and propagation of inspiratory neural activity as depolarizing wave dynamics on the entire transverse plane as well as within the preBötC. Novel combination of pharmacological blockade of glutamatergic transmission and mathematical subtraction of the video images under blockade from the control images enabled to extract glutamatergic signal propagations. By ultra-high-speed voltage-imaging we first demonstrated the inter-preBötC conduction process of inspiratory action potentials. Intra-preBötC imaging with high spatiotemporal resolution during a single spontaneous inspiratory cycle unveiled deterministic nonlinearities, i.e., chaos, in the population recruitment. Collectively, we comprehensively elucidated the anatomical pathways to and from the preBötC and dynamics of inspiratory neural information propagation: (1) From the preBötC in one side to the contralateral preBötC, which would synchronize the bilateral rhythmogenic kernels, (2) from the preBötC directly to the bilateral hypoglossal premotor and motor areas as well as to the nuclei tractus solitarius, and (3) from the hypoglossal premotor areas toward the hypoglossal motor nuclei. The coincidence of identified anatomical and functional connectivity between the preBötC and other regions in adult and neonatal rats, respectively, indicates that this fundamental connectivity is already well developed at the time of birth.

Botzinger expiratory-augmenting neurons and the parafacial respiratory group.

In neonatal rat brains in vitro, the rostral ventral respiratory column (rVRC) contains neurons that burst just before the phrenic nerve discharge (PND) and rebound after inspiration (pre-I neurons). These neurons, called parafacial respiratory group (pfRG), have been interpreted as a master inspiratory oscillator, an expiratory rhythm generator or simply as neonatal precursors of retrotrapezoid (RTN) chemoreceptor neurons. pfRG neurons have not been identified in adults, and their phenotype is unknown. Here, we confirm that the rVRC normally lacks pre-I neurons in adult anesthetized rats. However, we show that, during hypercapnic hypoxia, a population of rVRC expiratory-augmenting (E-AUG) neurons consistently develops a pre-I discharge. These cells reside in the Bötzinger region of the rVRC, they express glycine-transporter-2, and their axons arborize throughout the VRC. Hypoxia triggers an identical pre-I pattern in retroambigual expiratory bulbospinal neurons, but this pattern is not elicited in Bötzinger expiratory-decrementing neurons, Bötzinger inspiratory neurons, RTN neurons, and blood pressure-regulating neurons. In conclusion, under hypoxia in vivo, abdominal expiratory premotor neurons of adult rats develop a pre-I pattern reminiscent of that observed in neonate brainstems in vitro. In the rVRC of adult rats, pre-I cells include selected rhythmogenic neurons (glycinergic Bötzinger neurons) but not RTN chemoreceptors. We suggest that the pfRG may not be an independent rhythm generator but a heterogeneous collection of E-AUG neurons (glycinergic Bötzinger neurons, possibly facial motor and premotor neurons), the discharge of which becomes preinspiratory under specific experimental conditions resulting from, in part, a prolonged and intensified activity of postinspiratory neurons.

Substance P and enkephalinergic synapses onto neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats.

Our previous studies have demonstrated that neurokinin-1 receptor (NK1R)-immunoreactive (ir) neurons in the pre-Bötzinger Complex (pre-BötC), the hypothesized kernel of respiratory rhythmogenesis, receive both glutamatergic excitatory and GABAergic or glycinergic inhibitory inputs. Neuromodulators, such as substance P (SP) and opioids, play important roles in normal respiratory activity and respiratory disorders. The identification of the relationship between neurotransmitters and NK1R-ir neurons at the cellular level is essential for understanding the synaptic interaction within the pre-BötC network. Using immunofluorescence and immunogold-silver staining, we wished to exploit SP and enkephalin (ENK) immunoreactivity and their relationships with glutamate, GABA, glycine, or NK1R in the pre-BötC in adult Sprague-Dawley rats. The pre-BötC contained a substantial amount of SP-ir and ENK-ir boutons. They were largely colocalized with glutamate and much less so with GABA. Glycine immunoreactivity was rarely found in either SP-ir or ENK-ir boutons. A number of SP-ir boutons were ENK-ir as well. Synapses were commonly found between SP-ir or ENK-ir terminals and NK1R-ir neurons in the pre-BötC. Most of them were asymmetric. Symmetric synapses made up 10% of all synapses examined between SP-ir boutons and NK1R-ir neurons, and 19% of ENK/NK1R synapses. Colocalization of SP and/or ENK with glutamate in boutons in the pre-BötC implies the combined synaptic release of excitatory amino acid and neuropeptides, which may exert combined post-synaptic effects onto NK1R-ir neurons and contribute to respiratory activity.

GABAergic and glycinergic synapses onto neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats: light and electron microscopic studies.

The pre-Bötzinger complex (preBötC) in the ventrolateral medulla is thought to be the kernel for respiratory rhythm generation. Neurons in the preBötC contain intense neurokinin-1 receptor (NK1R) immunoreactivity. Some of these neurons in the adult preBötC are presumed to be the pre-inspiratory interneurons that are essential for generating respiratory rhythm in the neonate. Chloride-mediated synaptic inhibition is critical for rhythmogenesis in the adult. The present study used immunofluorescence histochemistry and immunogold-silver staining to determine the inhibitory synaptic relationship between glutamic acid decarboxylase (GAD)- or glycine transporter 2 (GlyT2)-immunoreactive (ir) boutons and NK1R-ir neurons in the preBötC of adult rats. Under the confocal microscope, we found that GAD- and GlyT2-ir boutons were in close apposition to NK1R-ir somas and dendrites in the preBötC. Under the electron microscope, GAD- and GlyT2-ir terminals were in close apposition to NK1R-ir somas and dendrites. Symmetric synapses were identified between GAD- or GlyT2-ir terminals and NK1R-ir neurons. A total of 51.6% GAD-ir and 38.2% GlyT2-ir terminals were found to contact or make synapses with NK1R-ir profiles, respectively. GAD- and GlyT2-ir terminals synapsed not only upon NK1R-ir neurons but also upon NK1R immuno-negative neurons. NK1R-ir neurons received both symmetric (presumed inhibitory) and asymmetric (presumed excitatory) synapses. Thus, the present findings provide the morphological basis for inhibitory inputs to NK1R-ir neurons in the preBötC, consistent with the suggestion that chloride-mediated synaptic inhibition may contribute importantly to rhythm generation by controlling the membrane potential trajectory and resetting rhythmic bursting of the kernel neurons in the adult.

Monosynaptic projections from the lateral periaqueductal gray to the nucleus retroambiguus in the rhesus monkey: implications for vocalization and reproductive behavior.

The periaqueductal gray (PAG) is known to be essential for vocalization and reproductive behavior. The PAG controls components of these behaviors by means of projections to the nucleus retroambiguus (NRA), a group of premotor neurons in the caudal medulla oblongata. In the accompanying study (VanderHorst et al., 2000 [accompanying study]), the NRA and its lumbosacral projections have been identified in the rhesus monkey. The present light and electron microscopical tracing study describes the PAG-NRA pathway in primates. To locate midbrain neurons projecting to the NRA, wheat germ agglutinin horseradish peroxidase (WGA-HRP) was injected into the NRA in six monkeys. To determine the distribution pattern of PAG axons in the medulla oblongata, WGA-HRP was injected into the PAG and adjacent tegmentum in three additional monkeys. In one of these three monkeys, biotinylated dextran amine and cholera toxin subunit b were injected into the lumbosacral cord to retrogradely identify NRA neurons. The results show that a compact group of neurons in the medial part of the lateral PAG at the intercollicular level sends a dense projection to the NRA. The projection is bilateral with a clear ipsilateral predominance. At the ultrastructural level, there are monosynaptic contacts between PAG fibers and NRA neurons, including NRA neurons that project to the lumbosacral cord. The synaptic contacts were primarily asymmetrical and the labeled terminal profiles contained spherical and dense core vesicles. It is concluded that there exists a strong and direct PAG-NRA pathway in the rhesus monkey. Because NRA neurons projecting to the lower lumbar cord are included, the PAG-NRA projection is likely to be involved not only in vocalization but also in other behaviors, such as receptive posture.

Scanning electron microscopy of the floor of the fourth ventricle in rats subjected to graded impact injury to the sensorimotor cortex.

OBJECT: Respiratory dysfunction including apnea frequently follows head injury in humans. The purpose of this study was to identify any structural alterations in the region of brainstem respiratory nuclei that might account for immediate postinjury respiratory abnormalities in anesthetized experimental animals. METHODS: Using scanning electron microscopy, the authors examined the floor of the fourth ventricle in injured rats after a piston strike to the sensorimotor cortex that depressed the dura 1, 2, or 4 mm. The rats were killed within minutes of injury. Cortical impact depths measuring either 1 or 2 mm (eight rats) produced no respiratory abnormalities, and the structural integrity of the ependymal lining of the ventricular floor in these animals was not compromised. Thirteen rats were subjected to impact to a 4-mm depth and 10 of these exhibited immediate temporary or permanent apnea. The medullae of nine of these rats were studied using scanning electron microscopy, and the fourth ventricular floors of all nine rats showed tears. Four rats that exhibited immediate, permanent apnea had tears in the caudal fourth ventricle floor near the obex, whereas five rats with no or only transient apnea had tears located more anteriorly, near the aqueduct or laterally. Changes in cerebrospinal fluid flow or pressure dynamics may have caused these tears. Light microscopy, focused near the area postrema, revealed a shearing defect through the ependyma of the fourth ventricular floor into the subjacent neuropil with a disruption of axonal pathways. CONCLUSIONS: Respiratory neuronal network components lying within 2 mm of the area postrema may well have been disrupted by the caudal tears producing permanent apnea. A similar phenomenon could account for the transient or permanent postinjury apnea seen in humans with severe head injury.