Testing the role of preBötzinger Complex somatostatin neurons in respiratory and vocal behaviors

Identifying neurons essential for the generation of breathing and related behaviors such as vocalisation is an important question for human health. The targeted loss of preBötzinger Complex (preBötC) glutamatergic neurons, including those that express high levels of somatostatin protein (SST neurons), eliminates normal breathing in adult rats. Whether preBötC SST neurons represent a functionally specialised population is unknown. We tested the effects on respiratory and vocal behaviors of eliminating SST neuron glutamate release by Cre‐Lox‐mediated genetic ablation of the vesicular glutamate transporter 2 (VGlut2). We found the targeted loss of VGlut2 in SST neurons had no effect on viability in vivo, or on respiratory period or responses to neurokinin 1 or μ‐opioid receptor agonists in vitro. We then compared medullary SST peptide expression in mice with that of two species that share extreme respiratory environments but produce either high or low frequency vocalisations. In the Mexican free‐tailed bat, SST peptide‐expressing neurons extended beyond the preBötC to the caudal pole of the VII motor nucleus. In the naked mole‐rat, however, SST‐positive neurons were absent from the ventrolateral medulla. We then analysed isolation vocalisations from SST‐Cre;VGlut2^F/F mice and found a significant prolongation of the pauses between syllables during vocalisation but no change in vocalisation number. These data suggest that glutamate release from preBötC SST neurons is not essential for breathing but play a species‐ and behavior‐dependent role in modulating respiratory networks. They further suggest that the neural network generating respiration is capable of extensive plasticity given sufficient time.     

Neurokinin-1 receptor-immunoreactive neurons of the ventral respiratory group in the rat

The rostral end of the ventral respiratory group (VRG) contains neurons that are intensely neurokinin-1 receptor (NK1R) immunoreactive (ir). It has been theorized that some of these cells might be critical to respiratory rhythmogenesis (Gray et al. [1999] Science 286:1566-1568). In the present study we determined what major transmitter these NK1R-ir cells make and whether they are bulbospinal or propriomedullary. NK1R-ir neurons were found in the VRG between Bregma levels -11.7 and -13.6 mm. The highest concentration was found between Bregma -12.3 and -13.0 mm. This region overlaps with the pre-B?tzinger complex (pre-B?tC) as it was found to contain many pre-inspiratory neurons, few E2-expiratory neurons, and no I-incremental neurons. VRG NK1R-ir neurons contain neither tyrosine hydroxylase (TH) nor choline acetyl-transferase (ChAT) immunoreactivity, although dual-labeled neurons were found elsewhere within the rostral medulla. GAD67 mRNA was commonly detected in the ventrolateral medulla (VLM) but rarely in the NK1R-ir neurons of the pre-B?tC region (6 % of somatic profiles). GlyT2 mRNA was commonly found in the pre-B?tC region but rarely within NK1R-ir neurons (1.3 %). Up to 40% of VRG NK1R-ir neurons were retrogradely labeled by Fluoro-Gold (FG) injected in the contralateral pre-B?tC region. Some NK1R-ir VRG neurons located caudal to Bregma -12.6 mm were retrogradely labeled by FG injected in the spinal cord (C4-C5, T2-T4). In sum, NK1R immunoreactivity is present in many types of ventral medullary neurons. Within the VRG proper, NK1R-ir neurons are concentrated in an area that overlaps with the pre-B?tC. Within this limited region of the VRG, NK1R-ir neurons are neither cholinergic nor catecholaminergic, and very few are gamma-aminobutyric acid (GABA)ergic or glycinergic. The data suggest that most NK1R-ir neurons of the pre-B?tC region are excitatory. Furthermore, the more rostral NK1R-ir cells are propriomedullary, whereas some of the caudal ones project to the spinal cord.     

Mu-opioid receptors are present in functionally identified sympathoexcitatory neurons in the rat rostral ventrolateral medulla

Agonists of the mu-opioid receptor (MOR) produce profound hypotension and sympathoinhibition when microinjected into the rostral ventrolateral medulla (RVL). These effects are likely to be mediated by the inhibition of adrenergic and other presympathetic vasomotor neurons located in the RVL. The present ultrastructural studies were designed to determine whether these vasomotor neurons, or their afferents, contain MORs. RVL bulbospinal barosensitive neurons were recorded in anesthetized rats and filled individually with biotinamide by using a juxtacellular labeling method. Biotinamide was visualized by using a peroxidase method and MOR was identified by using immunogold localization of an antipeptide antibody that recognizes the cloned MOR, MOR1. The subcellular relationship of MOR1 to RVL neurons with fast- or slow-conducting spinal axons was examined by electron microscopy. Fast- and slow-conducting cells were not morphologically distinguishable. Immunogold-labeling for MOR1 was found in all RVL bulbospinal barosensitive neurons examined (9 of 9). MOR1 was present in 52% of the dendrites from both types of cells and in approximately half of these dendrites the MOR1 was at nonsynaptic plasmalemmal sites. A smaller portion of biotinamide-labeled dendrites (16%) from both types of cells were contacted by MOR1-containing axons or axon terminals. Together, these results suggest that MOR agonists can directly influence the activity of all types of RVL sympathoexcitatory neurons and that MOR agonists may also influence the activity of afferent inputs to these cells. The heterogenous distribution of MORs within individual RVL neurons indicates that the receptor is selectively targeted to specific pre- and postsynaptic sites.     

Inhibition of protein kinase A activity depresses phrenic drive and glycinergic signalling,but not rhythmogenesis in anaesthetized rat

The cAMP–protein kinase A (PKA) pathway plays a critical role in regulating neuronal activity. Yet, how PKA signalling shapes the population activity of neurons that regulate respiratory rhythm and motor patterns in vivo is poorly defined. We determined the respiratory effects of focally inhibiting endogenous PKA activity in defined classes of respiratory neurons in the ventrolateral medulla and spinal cord by microinjection of the membrane‐permeable PKA inhibitor Rp‐adenosine 3′,5′‐cyclic monophosphothioate (Rp‐cAMPS) in urethane‐anaesthetized adult Sprague Dawley rats. Phrenic nerve activity, end‐tidal CO₂ and arterial pressure were recorded. Rp‐cAMPS in the preBötzinger complex (preBötC) caused powerful, dose‐dependent depression of phrenic burst amplitude and inspiratory period. Rp‐cAMPS powerfully depressed burst amplitude in the phrenic premotor nucleus, but had no effect at the phrenic motor nucleus, suggesting a lack of persistent PKA activity here. Surprisingly, inhibition of PKA activity in the preBötC increased phrenic burst frequency, whereas in the Bötzinger complex phrenic frequency decreased. Pretreating the preBötC with strychnine, but not bicuculline, blocked the Rp‐cAMPS‐evoked increase in frequency, but not the depression of phrenic burst amplitude. We conclude that endogenous PKA activity in excitatory inspiratory preBötzinger neurons and phrenic premotor neurons, but not motor neurons, regulates network inspiratory drive currents that underpin the intensity of phrenic nerve discharge. We show that inhibition of PKA activity reduces tonic glycinergic transmission that normally restrains the frequency of rhythmic respiratory activity. Finally, we suggest that the maintenance of the respiratory rhythm in vivo is not dependent on endogenous cAMP–PKA signalling.     

Glutamatergic neuronal projections from the marginal layer of the rostral ventral medulla to the respiratory centers in rats

The marginal layer (ML) that lines the ventral surface of the medulla oblongata (VMS) contains neurons thought to contribute to central chemoreception, the process by which systemic hypercapnia activates respiration. The transmitters and connectivity of ML neurons are poorly known. The present study focuses on a group of nonserotonergic ML neurons, often located in close proximity to the entry point of penetrating blood vessels. These neurons (approximately 300/brain) contain vesicular glutamate transporter2 (VGLUT2) mRNA and are thus probably glutamatergic. They cluster below the caudal half of the facial motor nucleus, lateral to the serotonergic cells of the ML. The projections of serotonergic and nonserotonergic ML neurons were investigated by retrograde labeling with Fluoro-Gold. ML VGLUT2 mRNA-expressing neurons lack spinal projections and innervate the dorsolateral pons and the ipsilateral ventral respiratory column (VRC), most particularly, the region of the pre-B?tzinger complex and rVRG. The latter two regions receive a very small input from ML serotonergic neurons which, instead, heavily innervate the spinal cord. In conclusion, a small region of the VMS marginal layer contains glutamatergic neurons that innervate the main respiratory centers of the medulla oblongata and pons. These glutamatergic neurons are located in a chemosensitive region of the ML and their projections are consistent with a role in central chemoreception. The serotonergic neurons of the ML, though known to be activated by CO(2), probably do not contribute to central chemoreception, given that they innervate sympathetic efferents and project at best very lightly to the VRC.     

Somatostatin is not co-localized in cholinergic neurons innervating the rat cerebral cortex-hippocampal formation

The somatostatin content and choline acetyltransferase activity of the rat cerebral cortex and hippocampus were examined after lesions to the nucleus basalis, the fornix and the dorsal hippocampus. Lesions of the nucleus basalis caused reductions in cortical choline acetyltransferase activity but had no effect on the concentration of somatostatin. Fornix transection caused a reduction in choline acetyltransferase activity in the dorsal hippocampus, but again was without effect on the somatostatin content. Excitotoxin lesions of the dorsal hippocampus caused a 69% reduction in somatostatin concentration in this structure, with no reduction in choline acetyltransferase activity. The results indicate that somatostatin and choline acetyltransferase are in separate populations of neurons in the rat cerebral cortex and hippocampus.     

Morphology of augmenting inspiratory neurons of the ventral respiratory group in the cat

The present study examined, in Nembutal-anesthetized and artificially ventilated cats, the morphologic properties of the inspiratory neurons of the ventral respiratory group (VRG). Horseradish peroxidase (HRP) was injected into 21 augmenting inspiratory or late inspiratory neurons with peak firing rates in the late inspiratory phase. The majority of the stained neurons were antidromically activated by stimulation of the cervical cord. Thirteen somata, located within or around the nucleus ambiguus (AMB), between 100 microns caudally and 2,000 microns rostrally to the obex, were stained. In ten cases, the stem axons issuing from the cells of origin coursed medially to cross the midline without giving off any axonal collaterals. Three neurons gave rise to axonal collaterals on the ipsilateral side, distributing boutons in the medullary reticular formation, in the vicinity of the AMB, hypoglossal nucleus, solitary tract, and dorsal motor nucleus of the vagus. In eight neurons, only the axons were labeled; in four of these, which were antidromically activated from the spinal cord, the stem axons crossed the midline 2,000-3,000 microns rostral to the obex and descended in the reticular formation around the AMB down to the cervical cord. They issued several axonal collaterals, distributing terminal boutons at the level of the caudal end of the retrofacial nucleus and about 1,000 microns rostral and caudal from the obex. Terminals were found mainly in and around the AMB, and a few were found in the vicinity of the dorsal motor nucleus of the vagus. The remaining four nonactivated axons distributed their terminal boutons widely in the reticular formation around the AMB. Thus, the augmenting inspiratory neurons of the VRG were shown to project not only to the spinal cord, but also to the VRG, hypoglossal nucleus, and dorsal motor nucleus of the vagus.     

Firing patterns of pre-Bötzinger and Bötzinger neurons during hypocapnia in the adult rat

Controversy exists about how a coordinated respiratory rhythm is generated in the brainstem. Some authors suggest that neurons in the pre-Bötzinger complex are key to initiation of all types of breathing. While, on the other hand, it has been reported that some pre-Bötzinger neurons fail to maintain a rhythmic discharge in phase with phrenic nerve discharge during mechanical hyperventilation. Extracellular recordings were made from respiratory units in the pre-Bötzinger and Bötzinger complexes of 13 anaesthetised, paralysed and vagotomised rats. Central respiratory activity was monitored from the C5 phrenic nerve. During mechanical hyperventilation, several changes were observed in the phrenic neurogram. Firstly, the frequency and amplitude of integrated phrenic nerve discharge were reduced and reversibly stopped. Secondly, the patterned discharges changed from an augmenting to a variety of non-augmenting patterns in 53 of 60 cases. In some cases (n=9) we observed that the pattern appeared to have two components, an early short duration discharge followed by a longer duration discharge. Respiratory units also started to show different firing patterns during mechanical hyperventilation. In general, they were divided into those units that fired tonically (n=28) and units that became silent (n=32), before phrenic nerve discharge ceased coincidently with complete apnoea. Of particular interest were those expiratory–inspiratory units in the pre-Bötzinger complex (n=8) that narrowed their firing period towards late expiration and early inspiration during mechanical hyperventilation. Given their firing features, it is possible that these expiratory–inspiratory units may participate in generation of the early inspiratory component of phrenic nerve discharge.     

Medullary projection of nonaugmenting inspiratory neurons of the ventrolateral medulla in the cat.

In Nembutal-anesthetized, immobilized, and artificially ventilated cats, we studied the morphological characteristics of inspiratory neurons with nonaugmenting firing patterns. HRP was injected intracellularly into a total of 22 neurons of the B?tzinger complex (BOT) and the ventral respiratory group (VRG). In 20 cases somata with their axonal trajectories were stained, and in two cases only axons were stained. None of the neurons stained could be antidromically activated by stimulation of the cervical cord. The somata of 20 neurons were located in the vicinity of the nucleus ambiguus or the retrofacial nucleus (RFN) between 600 microns and 2,800 microns caudal to the rostral end of the RFN. Their axons could be traced for a distance of several millimeters on the side of the somata, and showed various projection patterns. According to these projection patterns, the 20 neurons were tentatively classified into four groups: A (8/20), B (4/20), C (6/20), and motoneurons (2/20). Group A neurons gave off extensive axon collaterals that arborized and distributed boutons predominantly in the BOT and the VRG areas. Group B neurons had less extensive axon collaterals with various projection patterns, projecting rarely to the BOT or the VRG area. Group C neurons sent their stem axons, without issuing any axonal collaterals, to the contralateral side in five cases and to the ipsilateral pons in one case. The two motoneurons had axons leaving the brainstem without any intramedullary collaterals. Thus, the nonaugmenting inspiratory neurons showed morphological variations, which may play different roles in neural control of respiration.     

Somatostatin produces apnea and is localized in medullary respiratory nuclei: a possible role in apneic syndromes

Immunocytochemical studies on the nucleus of the tractus solitarius and adjacent areas of the dorsal medulla of the rat demonstrate the existence of somatostatin immunoreactive nerve cell bodies and nerve terminals within the ventrolateral and ventral subnuclei of the nucleus of the tractus solitarius. Injections of somatostatin (6 nmol in μl) into the cisterna magna of chloralose-anesthetized rats produced an appea with a latency of 5–7 min. This apnea was preceded by slow deep brething, a reduction in heart rate and fall of arterial blood pressure. The apnea was usuallly irreversible leading to death of the animal. These respiratory and cardiovascular effects of somatostatin were not abolished either by bilateral vagotomy or by low decerebration (below the inferior colliculus). It is suggested that activation of somatostatin receptors linked to neurons in medullary respiratory nuclei might be responsible for the inhibition of respiratory neuronal activity and thus may mediate apneic conditions.     

Localization of respiratory bulbospinal and propriobulbar neurons in the region of the nucleus ambiguus of the rat

The location of neurons within the ventral respiratory group (VRG) of rat was mapped following injections of 3 different fluorochrome tracers into different sites known to receive projections from VRG neurons. Injection sites included muscles innervated by the vagus (X) and glossopharyngeal (IX) nerves, and the sites of expiratory activity in the caudal medulla and of inspiratory activity in the spinal cord at the C4 level. Labeling of vagal motoneurons resulting from fluorochrome injections into muscles innervated by X and IX nerves was always ipsilateral to the site of injection. Both propriobulbar and bulbospinal neurons had primarily ipsilateral projections. No double-labeled cell bodies were observed. The cell bodies of the 3 types of neurons, propriobulbar, bulbospinal and vagal/glossopharyngeal, were unevenly distributed along the rostrocaudal axis of the VRG, suggesting a complex mosaic of neurons which regulate respiratory-related functions such as swallowing and vocalization.     

Characteristics of midbrain respiratory neurons in sleep and wakefulness in the cat

Midbrain neurons were recorded in sleep and wakefulness in chronic cats. In the first phase of this study, we attempted to detect respiratory neurons by observing neuronal activity on an oscilloscope and listening to it after audioamplification. We studied 780 neurons in this non-quantitative way and failed to detect any respiratory activity. In the second phase, 203 neurons were analyzed statistically: 15% of these had activity patterns significantly related to the respiratory cycle. In the third and final phase, we studied the details of the activity of midbrain respiratory neurons. Sixteen of 281 single neurons had discharge patterns significantly related to the respiratory cycle, but only 3 of the 16 were related to breathing with a consistency that essentially excluded the possibility of a false positive error. All 3 of these cells were only intermittently respiratory, and the intermittency varied from cell to cell and tended to depend upon the state of consciousness.These results differ substantially from reports that more than 30% of midbrain and diencephalic cells are respiratory neurons. The differences may be explained by the previous authors' use of a statistic, the respiratory modulation index, that, as shown here and in a previous simulation study, produces a large number of false positive errors.     

Somatostatin-like neurons are expressed in fetal neocortical homografts in adult rat spinal cord

Fetal central nervous system homografts to adult spinal cord are considered a potential aid for recovery of function after paraplegia. This study utilizes somatostatin (SOM) immunohistochemistry to study the organization of an embryonic day 14 (E14) neocortical homograft into the spinal cord of an adult host over 6 postoperative months. Although the E14 homograft does not contain SOM-positive cells, SOM-reactive neurons are expressed by 30 days postimplantation and are still present in 6-month-old homografts. SOM-immunoreactive neurons are bitufted or multipolar and have dendrites that are confined to the graft. The homograft contains SOM-immunoreactive axons entering and/or exiting from lamina II in the host dorsal horn and SOM-positive homografted neurons send axons into the host ventral columns. These data show that the SOM peptide neocortical phenotype is preserved in homografts to spinal cord but there is anatomical host-homograft integration.     

Dopaminergic neurons in the rat ventral tegmental area. III. Effects of -and -amphetamine

By use of various histochemical techniques, it was shown that both DA and non-DA cells in the VTA project to the NAc. Of these VTA-NAc output cells, the great majority were DA-containing cells. A small number of non-DA cells were encountered most frequently in the lateral part of the VTA. Correspondingly, two distinct groups of neurons, types I and II, could be identified by antidromic stimulation of the NAc. Several lines of evidence suggest that type I cells are DA-containing neurons. The evidence may be summarized as follows:

1. (1) type I cells had a slow-bursting or regular firing pattern, slow discharge rate and wide spike duration which appears to be identical to the characteristics of DA neurons originally described by Bunney et al.¹⁶;

2. (2) the great majority of these cells could be activated antidromically by stimulation of the NAc;

3. (3) the conduction velocity and absolute refractory period of type I cells are consistent with unmyelinated fine DA fibers;

4. (4) injection of 6-OHDA, but not 5,7-DHT directly in the MFB blocked antidromic responses of these cells;

5. (5) they were extremely sensitive to intravenously administered DA agonist apomorphine (ID₅₀ = 7 μg/kg); and

6. (6) direct fluorescence histochemical examination of serial sections from brains of animals in which type I cells have been identified by antidromic stimulation of the NAc showed that type I cells are most likely catecholamine-containi ng neurons. By contrast, type II cells possessed an entirely different spectrum of physiological characteristics; in addition, they showed no consistent response to apomorphine and their antidromic responses to stimulation of the NAc were not affected by 6-OHDA. It is concluded that (1) VTA output neurons consist of both DA and nonDA neurons, and (2) identified types I and II neurons in the VTA by antidromic stimulation of the NAc are DA and non-DA cells, respectively.

A cross-correlation study of interactions among respiratory neurons of dorsal, ventral and retrofacial groups in cat medulla

In anesthetized or decerebrated cats, extracellular activities of pairs of respiratory neurons located in the regions of the dorsal (DRN), ventral (VRN) and retrofacial (RFN) medullary respiratory nuclei were recorded using two separate microelectrodes. Neurons were classified as bulbospinal or laryngeal if stimulation of the spinal cord or vagus nerve elicited antidromic action potentials, or as propriobulbar if they were not antidromically activated. Of 163 pairs of single unit activities, either inspiratory (143 pairs) or expiratory (20 pairs), cross-correlation analyses indicated that 23% had short latency peaks, either broad (12%) or sharp (1%) in their cross-correlograms, 3% had short latency troughs and 74% had flat cross-correlograms. When the two neurons were located in the DRN (68 pairs) the probability of obtaining a positive cross-correlogram was high for inspiratory bulbospinal neurons, indicating shared inputs and excitatory relationships. When one neuron of the pair was located in the RFN and the other in either the DRN or VRN (95 pairs), cross-correlation analysis revealed shared inputs, excitatory and inhibitory relationships. Among expiratory neurons interactions were only inhibitory with a more frequent incidence (320) than between inspiratory neurons (2143. Our results indicate that: (i) short time scale synchrony due to shared inputs (broad peaks) are largely distributed in the respiratory neuronal network and operate over long distance (i.e. RFN, caudal medulla); (ii) excitatory coupling may exist between remote neurons but is more frequent between inspiratory bulbospinal neurons located in the DRN.     

Synaptic contacts of vesicular glutamate transporter 2 fibres on chemically identified neurons of the hypothalamic suprachiasmatic nucleus of the rat

The hypothalamic suprachiasmatic nucleus (SCN), which plays a pivotal role in the control of circadian rhythms, consists of several neuronal subpopulations characterized by different neuroactive substances. This prominent cell group has a fairly rich glutamatergic innervation, but the cell types that are targeted by this innervation are unknown. Therefore, the purpose of the present study was to examine the relationship between the afferent glutamatergic axon terminals and the vasoactive intestinal polypeptide (VIP)-, arginine-vasopressin (AVP)- and gamma-aminobutyric acid (GABA)-positive neurons of the SCN. Glutamatergic elements were revealed via immunocytochemical double-labelling for vesicular glutamate transporter type 1 (VGluT1) and type 2 (VGluT2), and brain sections were imaged via confocal laser-scanning microscopy and electron microscopy. Numerous VGluT2-immunoreactive axons were observed to be in synaptic contact with VIP- and GABA-positive neurons, and only a few synapses were detected between VGluT2 boutons and AVP neurons. VGluT1 axon terminals exhibiting very moderate distribution in this cell group were observed to be in synaptic contact with chemically unidentified neurons. The findings provide the first morphological data on the termination of presumed glutamatergic fibres on chemically identified neurons of the rat SCN, and indicate that all three prominent cell types of the cell group receive glutamatergic afferents.     

Stimulation of the midbrain periaqueductal gray modulates preinspiratory neurons in the ventrolateral medulla in the rat in vivo

The midbrain periaqueductal gray (PAG) is involved in many basic survival behaviors that affect respiration. We hypothesized that the PAG promotes these behaviors by changing the firing of preinspiratory (pre‐I) neurons in the pre‐Bötzinger complex, a cell group thought to be important in generating respiratory rhythm. We tested this hypothesis by recording single unit activity of pre‐Bötzinger pre‐I neurons during stimulation in different parts of the PAG. Stimulation in the dorsal PAG increased the firing of pre‐I neurons, resulting in tachypnea. Stimulation in the medial part of the lateral PAG converted the pre‐I neurons into inspiratory phase‐spanning cells, resulting in inspiratory apneusis. Stimulation in the lateral part of the lateral PAG generated an early onset of the pre‐I neuronal discharge, which continued throughout the inspiratory phase, while at the same time attenuating diaphragm contraction. Stimulation in the ventral part of the lateral PAG induced tachypnea but inhibited pre‐I cell firing, whereas stimulation in the ventrolateral PAG inhibited not only pre‐I cells but also the diaphragm, leading to apnea. These findings show that PAG stimulation changes the activity of the pre‐Bötzinger pre‐I neurons. These changes are in line with the different behaviors generated by the PAG, such as the dorsal PAG generating avoidance behavior, the lateral PAG generating fight and flight, and the ventrolateral PAG generating freezing and immobility. J. Comp. Neurol. 521: 3083–3098, 2013. © 2013 Wiley Periodicals, Inc.