Generation of eupnea and sighs by a spatiochemically organized inspiratory network.

The discovery of the rhythmogenic pre-Bötzinger complex (preBötC) inspiratory network, which remains active in a transverse brainstem slice, greatly increased the understanding of neural respiratory control. However, basic questions remain unanswered such as (1) What are the necessary and sufficient slice boundaries for a functional preBötC? (2) Is the minimal preBötC capable of reconfiguring between inspiratory-related patterns (e.g., fictive eupnea and sighs)? (3) How is preBötC activity affected by surrounding structures? Using newborn rat slices with systematically varied dimensions in physiological [K(+)] (3 mM), we found that a 175 microm thickness is sufficient for generating inspiratory-related rhythms. In 700-microm-thick slices with unilaterally exposed preBötC, a kernel <100 microm thick, centered 0.5 mm caudal to the facial nucleus, is necessary for rhythm generation. Slices containing this kernel plus caudal structures produced eupneic bursts of regular amplitude, whereas this kernel plus rostral tissue generated sighs, intermingled with eupneic bursts of variable amplitude ("eupnea-sigh pattern"). After spontaneous arrest of rhythm, substance-P or neurokinin-1 (NK1) receptor agonist induced the eupnea-sigh burst pattern in > or = 250-microm-thick slices, whereas thyrotropin-releasing hormone or phosphodiesterase-4 blockers evoked the eupnea burst pattern. Endogenous rhythm was depressed by NK1 receptor antagonism. Multineuronal Ca(2+) imaging revealed that preBötC neurons reconfigure between eupnea and eupnea-sigh burst patterns. We hypothesize a (gradient-like) spatiochemical organization of regions adjacent to the preBötC, such that a small preBötC inspiratory-related oscillator generates eupnea under the dominant influence of caudal structures or thyrotropin-releasing hormone-like transmitters but eupnea-sigh activity when the influence of rostral structures or substance-P-like transmitters predominates.

Role of somatostatin and apoptosis in breathing control in sudden perinatal and infant unexplained death.

OBJECTIVES: To obtain basic information about the expression of somatostatin in the human central nervous system and, in particular, to evaluate its possible involvement in unexplained perinatal and in sudden infant death syndrome. MATERIAL: Sixty-seven brainstems from subjects aged from 30 gestational weeks to 12 postnatal months, dying of both known and unknown causes, were selected for this study. The unexplained deaths included 17 sudden intrauterine deaths, 5 sudden neonatal deaths and 28 sudden infant deaths. METHOD: All brainstems were fixed in 10% phosphate-buffered formalin, processed and embedded in paraffin, according to our protocol available on the web site: http://users.unimi.it/-pathol/sids/riscontro_diagnostico_e.html. The distribution of the somatostatin in the brainstem was studied by immunohistochemistry on serial sections. RESULTS: We observed an intense somatostatin positivity in many brainstem nuclei prevalently involved in the respiratory activity (parabrachial/Kölliker-Fuse complex, locus coeruleus, hypoglossus nucleus, dorsal vagus motor nucleus, tractus solitarii nucleus, ambiguus nucleus, reticular formation) in stillbirths. In 10 fetuses with unexplained death the neurons of the hypoglossus nucleus were somatostatin-negative. In the postnatal deaths, we observed immunopositivity in the ventrolateral and ventral subnuclei of the tractus solitarii nucleus. Besides, in 15 sudden infant death victims and in 1 control case, somatostatin-positive neurons were also present in the hypoglossus nucleus. In 10 of these 15 cases, a high apoptotic index was also reported. CONCLUSIONS: We suggest that abnormalities in the distribution of SS in the hypoglossus nucleus before and after birth may contribute to the induction of both fatal breathing in prenatal life and abnormal ventilatory control after birth leading to irreversible apnea.

Cerebral blood flow sensitivity to CO2 measured with steady-state and Read's rebreathing methods.

The ventilatory response to carbon dioxide (CO2) measured by the steady-state method is lower than that measured by Read's rebreathing method. A change in end-tidal P CO2 (PET CO2) results in a lower increment change in brain tissue P CO2 (Pt CO2) in the steady-state than with rebreathing: since Pt(CO2) determines the ventilatory response to CO2, the response is lower in the steady-state. If cerebral blood flow (CBF) responds to Pt CO2, the CBF-CO2 response should be lower in the steady-state than with rebreathing. Six subjects undertook two protocols, (a) steady-state: PET CO2 was held at 1.5 mmHg above normal (isocapnia) for 10 min, then raised to three levels of hypercapnia, (8 min each; 6.5, 11.5 and 16.5 mmHg above normal, separated by 4 min isocapnia). End-tidal P O2 was held at 300 mmHg; (b) rebreathing: subjects rebreathed via a 6 L bag filled with 6.5% CO2 in O2. Transcranial Doppler-derived CBF yielded a higher CBF-CO2 sensitivity in the steady-state than with rebreathing, suggesting that CBF does not respond to Pt CO2.

Relationship between two types of vesicular glutamate transporters and neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats: light and electron microscopic studies.

Our previous study demonstrated GABAergic and glycinergic synapses onto neurokinin-1 receptor (NK1R)-immunoreactive (ir) neurons in the pre-Bötzinger complex (pre-BötC), the hypothesized kernel of normal respiratory rhythmogenesis. In the present study, we aimed to identify glutamatergic synapses onto NK1R-ir pre-BötC neurons, as excitatory synaptic transmission is a prerequisite to normal respiratory rhythmogenesis. Two types of vesicular glutamate transporters (VGLUT), VGLUT1 and VGLUT2, have been recently implicated in glutamate-mediated transmission. The present study used immunofluorescence and immunogold-silver staining to determine the relationship between the transporters and NK1R-ir neurons in the pre-BötC of adult rats. Under the confocal laser-scanning microscope, VGLUT2-ir boutons were found to be widely distributed in the pre-BötC, some of which were in close apposition to NK1R-ir somas and dendrites. VGLUT1-ir boutons were relatively rare and only a few were found to be in close apposition to NK1R-ir somas and dendrites. Electron microscopic observation revealed that approximately 41% of VGLUT2-ir terminals were in close apposition to, or made asymmetric synapses with NK1R-ir somas and dendrites in the pre-BötC. On the other hand, 50.5% of NK1R-ir dendrites were closely apposed to, or synapsed with VGLUT2-ir terminals. Occasionally, VGLUT1-ir terminals were found in close apposition to NK1R-ir somas or dendrites, but we were unable to identify synapses between them. The present findings provide the morphological basis for excitatory synaptic inputs onto NK1R-ir neurons in the pre-BötC. VGLUT2 may be involved in a dominant excitatory synaptic pathway for normal respiratory rhythmogenesis.

The case for the bulbospinal respiratory nitric oxide synthase-immunoreactive pathway in the dog.

Previous investigations from our laboratory have documented that the neuropil of the phrenic nucleus contains a dense accumulation of punctate nicotinamide adenine dinucleotide phosphate diaphorase staining. In this study we investigated the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the phrenic nucleus in C3-C5 segments, supposed to be the terminal field of the premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway in the dog. As the first step, nitric oxide synthase immunohistochemistry was used to characterize nitric oxide synthase-immunoreactive staining of the phrenic nucleus and nitric oxide synthase-containing neurons in the dorsal and rostral ventral respiratory group and in the Bötzinger complex of the medulla. Dense punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the phrenic nucleus. Several thin bundles of nitric oxide synthase-immunoreactive fibers were found to enter the phrenic nucleus from the lateral and ventral column. Nitric oxide synthase-containing neurons were revealed in the dorsal respiratory group of medulla corresponding to the ventrolateral nucleus of the solitary tract and in the rostral ventral respiratory group beginning approximately 1 mm caudal to the obex and reaching to 650 microm rostral to the obex. Axotomy-induced retrograde changes, consisting in a strong upregulation of nitric oxide synthase-containing neurons, were found in the dorsal and rostral ventral respiratory group contralateral to the hemisection performed at the C2-C3 level. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the phrenic nucleus ipsilaterally with the hemisection was detected, thus revealing that a crossed premotor bulbospinal respiratory pathway contains a fairly high number of nitric oxide synthase-immunopositive fibers terminating in the phrenic nucleus. The use of the retrograde fluorescent tracer Fluorogold injected into the phrenic nucleus and an analysis of sections cut through the dorsal and rostral ventral respiratory group and Bötzinger complex of medulla and processed for nitric oxide synthase immunocytochemistry revealed that approximately 73.8% of crossed premotor bulbospinal respiratory nitric oxide synthase-immunoreactive axons originate in the rostral ventral respiratory group and 26.2% is given by nitric oxide synthase-containing neurons of the dorsal respiratory group. A few premotor nitric oxide synthase-immunoreactive axons originating from the Bötzinger complex were found. In summary, the present study provides evidence for a hitherto unknown premotor bulbospinal respiratory nitric oxide synthase-immunoreactive pathway connecting the bulbar respiratory centers with the motor neurons of the phrenic nucleus in the dog.

Muscimol dialysis in the retrotrapezoid nucleus region inhibits breathing in the awake rat.

Under anesthesia, inactivation of the retrotrapezoid nucleus (RTN) region markedly inhibits breathing and chemoreception. In conscious rats, we dialyzed muscimol for 30 min to inhibit neurons of the RTN region reversibly. Dialysis of artificial cerebrospinal fluid had no effect. Muscimol (1 or 10 mM) significantly decreased tidal volume (VT) (by 16-17%) within 15 min. VT remained decreased for 50 min or more, with recovery by 90 min. Ventilation (VE) decreased significantly (by 15-20%) within 15 min and then returned to baseline within 40 min as a result of an increase in frequency. This, we suggest, is a compensatory physiological response to the reduced VT. Oxygen consumption was unchanged. In response to 7% CO(2) in the 1 mM group, absolute VE and change in VE were significantly reduced (by 19-22%). In the 10 mM group, the response to dialysis included a time-related increase in frequency and decrease in body temperature, which may reflect greater spread of muscimol. In the awake rat, the RTN region provides a portion of the tonic drive to breathe, as well as a portion of the response to hypercapnia.

Expression of N-methyl-D-aspartate receptor subunits in the rat parabrachial and Kölliker-Fuse nuclei and in selected pontomedullary brainstem nuclei.

In the present study, we investigated the expression of N-methyl-D-aspartate (NMDA) receptor subunits in the parabrachial/Kölliker-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.

A role for KATP+-channels in mediating the elevations of cerebral blood flow and arterial pressure by hypoxic stimulation of oxygen-sensitive neurons of rostral ventrolateral medulla.

Reticulospinal sympathoexcitatory neurons of rostral ventrolateral medulla (RVL) are selectively excited by hypoxia to elevate arterial pressure (AP) and cerebral blood flow (rCBF), that are elements of the oxygen-conserving (diving) reflex. We investigated whether KATP+-channels participate in this. Tolbutamide and glibenclamide, KATP+-channel blockers, microinjected into RVL in anesthetized rats, dose-dependently and site-specifically elevated AP and rCBF and potentiated responses to hypoxemia. KATP+-channels may mediate hypoxic excitation of oxygen-sensing RVL neurons.

Substance P receptors in brain stem respiratory centers of the rat: regulation of NK1 receptors by hypoxia.

Substance P (SP) is a key neurotransmitter involved in the brain stem integration of carotid body chemoreceptor reflexes. In this study, the characteristics and location of SP receptors in the rat brain stem and their regulation by hypoxia were investigated using homogenate radioligand binding and quantitative autoradiography. Specific binding of [125I] Bolton-Hunter SP (BHSP) to brain stem homogenates was saturable (approximately 0.3 nM) and to a single class of high-affinity sites (K(d), 0.16 nM; maximum density of binding sites, 0.43 fmol/mg wet weight tissue). The order of potency of agonists for inhibition of BHSP binding was SP > [Sar9Met(O2)11]SP >> neurokinin A > septide > neurokinin B >> [Nle10]-neurokinin A(4-10) = senktide, and for nonpeptide antagonists, RP 67580 > CP-96,345 >> RP 68651 = CP-96,344, consistent with binding to NK1 receptors. The effect of single and multiple, 5-min bouts of hypoxia (8.5% O2/91.5% N2) on BHSP binding was investigated using quantitative autoradiography. Binding sites were localized to the lateral, medial and commissural nucleus of the solitary tract (NTS), the hypoglossal nucleus, central gray and the spinal trigeminal tract and nucleus (Sp5 and nSp5, respectively). Five min after a single bout of hypoxia, the density of BHSP binding sites had decreased significantly (P < .05) in the medial NTS (-33%) and lateral NTS (-24%) when compared to normoxic controls. However, the normal receptor complement was restored within 60 min of the hypoxic challenge. In the Sp5, a significant decrease (P < .05) in binding was observed 5 min after hypoxia which was still apparent after 60 min. In contrast, the density of BHSP binding sites in the hypoglossal nucleus decreased slowly and was significantly lower (P < .05) than normoxic controls 60 min after hypoxia. Five min after repetitive hypoxia (3 x 5 min bouts), BHSP binding in the NTS was reduced by more than 40%. Studies in homogenates showed that the affinity of SP for BHSP binding sites was not affected by repetitive hypoxia (K(d)s, normoxic, 0.27 nM; hypoxic, 0.24 nM). These data suggest that afferent input from carotid body chemoreceptors may dynamically regulate NK1 receptors in several brain stem nuclei that are intimately involved in stimulating ventilation during hypoxia, and that the time-course of receptor turnover may differ from region to region in the brain stem. The temporary loss of NK1 receptors in the NTS may partly explain why adequate ventilation is often not maintained during hypoxia.