Ann M. Schreihofer

Identification of C1 presympathetic neurons in rat rostral ventrolateral medulla by juxtacellular labeling in vivo

The rostral ventrolateral medulla (RVLM) contains barosensitive, bulbospinal neurons that provide the main supraspinal excitatory input to sympathetic vasomotor preganglionic neurons. However, the phenotype of the critical RVLM cells has not been conclusively determined. The goal of the current study was to identify the proportion of electrophysiologically defined, putative, presympathetic RVLM neurons that are C1 cells. We used a juxtacellular labeling technique to individually fill spontaneously active, barosensitive, bulbospinal RVLM neurons with biotinamide following electrophysiological characterization in chloralose‐anesthetized rats. To determine whether these neurons could be classified as C1 cells, the biotinamide‐labeled cells were processed for detection of tyrosine hydroxylase. The majority of barosensitive bulbospinal RVLM neurons were tyrosine hydroxylase immunoreactive (TH‐ir; 28 of 39). All of the barosensitive bulbospinal RVLM neurons with axonal conduction velocities in the C fiber range (<1 m/second) were TH‐ir (n = 16), whereas faster conducting cells (1 to 7 m/second) were either lightly TH‐ir (n = 12) or not detectably TH‐ir (n = 11). Adjacent respiratory‐related RVLM units labeled with biotinamide were not detectably TH‐ir (n = 10). To verify that TH‐ir cells were indeed adrenergic, a subset of barosensitive bulbospinal cells labeled with biotinamide were examined for phenylethanolamine N‐methyltransferase immunoreactivity (PNMT‐ir). Three slowly conducting cells had detectable PNMT‐ir, and two fast‐conducting cells had no detectable PNMT‐ir. These results indicate that the majority of bulbospinal RVLM neurons with putative sympathoexcitatory function are C1 cells. J. Comp. Neurol. 387:524–536, 1997.

Vesicular glutamate transporter DNPI/VGLUT2 is expressed by both C1 adrenergic and nonaminergic presympathetic vasomotor neurons of the rat medulla

The main source of excitatory drive to the sympathetic preganglionic neurons that control blood pressure is from neurons located in the rostral ventrolateral medulla (RVLM). This monosynaptic input includes adrenergic (C1), peptidergic, and noncatecholaminergic neurons. Some of the cells in this pathway are suspected to be glutamatergic, but conclusive evidence is lacking. In the present study we sought to determine whether these presympathetic neurons express the vesicular glutamate transporter BNPI/VGLUT1 or the closely related gene DNPI, the rat homolog of the mouse vesicular glutamate transporter VGLUT2. Both BNPI/VGLUT1 and DNPI/VGLUT2 mRNAs were detected in the medulla oblongata by in situ hybridization, but only DNPI/VGLUT2 mRNA was present in the RVLM. Moreover, BNPI immunoreactivity was absent from the thoracic spinal cord lateral horn. DNPI/VGLUT2 mRNA was present in many medullary cells retrogradely labeled with Fluoro‐Gold from the spinal cord (T2; four rats). Within the RVLM, 79% of the bulbospinal C1 cells contained DNPI/VGLUT2 mRNA. Bulbospinal noradrenergic A5 neurons did not contain DNPI/VGLUT2 mRNA. The RVLM of six unanesthetized rats subjected to 2 hours of hydralazine‐induced hypotension contained tenfold more c‐Fos‐ir DNPI/VGLUT2 neurons than that of six saline‐treated controls. c‐Fos‐ir DNPI/VGLUT2 neurons included C1 and non‐C1 neurons (3:2 ratio). In seven barbiturate‐anesthetized rats, 16 vasomotor presympathetic neurons were filled with biotinamide and analyzed for the presence of tyrosine hydroxylase immunoreactivity and/or DNPI/VGLUT2 mRNA. Biotinamide‐labeled neurons included C1 and non‐C1 cells. Most non‐C1 (9/10) and C1 presympathetic cells (5/6) contained DNPI/VGLUT2 mRNA. In conclusion, DNPI/VGLUT2 is expressed by most blood pressure‐regulating presympathetic cells of the RVLM. The data suggest that these neurons may be glutamatergic and that the C1 adrenergic phenotype is one of several secondary phenotypes that are differentially expressed by subgroups of these cells. J. Comp. Neurol. 444:207–220, 2002.

The Baroreflex And Beyond: Control Of Sympathetic Vasomotor Tone By Gabaergic Neurons In The Ventrolateral Medulla

1. Barosensitive, bulbospinal neurons in the rostral ventrolateral medulla (RVLM), which provide the major tonic excitatory drive to sympathetic vasomotor neurons, are prominently inhibited by GABA.

Regulation of sympathetic tone and arterial pressure by rostral ventrolateral medulla after depletion of C1 cells in rat

1 In this study we examined whether the rostral ventrolateral medulla (RVLM) maintains resting sympathetic vasomotor tone and activates sympathetic nerve activity (SNA) after the depletion of bulbospinal C1 adrenergic neurones. 2 Bulbospinal C1 cells were destroyed (≈84% loss) by bilateral microinjections (spinal segments T2‐T3) of an anti‐dopamine‐β‐hydroxylase antibody conjugated to the ribosomal toxin saporin (anti‐DβH‐SAP). 3 Extracellular recording and juxtacellular labelling of bulbospinal barosensitive neurones in the RVLM revealed that treatment with anti‐DβH‐SAP spared the lightly myelinated neurones with no tyrosine hydroxylase immunoreactivity. 4 In rats treated with anti‐DβH‐SAP, inhibition of RVLM neurones by bilateral microinjection of muscimol eliminated splanchnic SNA and produced the same degree of hypotension as in control rats. 5 Following treatment with anti‐DβH‐SAP the sympathoexcitatory (splanchnic nerve) and pressor responses to electrical stimulation of the RVLM were reduced. 6 Treatment with anti‐DβH‐SAP also eliminated the majority of A5 noradrenergic neurones. However, rats with selective lesion of A5 cells by microinjection of 6‐hydroxydopamine into the pons showed no deficits to stimulation of the RVLM. 7 In summary, the loss of 84% of bulbospinal adrenergic neurones does not alter the ability of RVLM to maintain SNA and arterial pressure at rest in anaesthetized rats, but this loss reduces the sympathoexcitatory and pressor responses evoked by RVLM stimulation. The data suggest sympathoexcitatory roles for both the C1 cells and non‐C1 cells of the RVLM and further suggest the C1 cells are critical for the full expression of sympathoexcitatory responses generated by the RVLM.

Evidence for glycinergic respiratory neurons: Bötzinger neurons express mRNA for glycinergic transporter 2

Bötzinger (BÖTZ) neurons in the rostral ventrolateral medulla fire during the late expiratory phase of the respiratory cycle. These cells inhibit phrenic motor neurons and several types of respiratory neurons in the medulla oblongata. BÖTZ cells produce a fast, chloride‐mediated inhibition of their target neurons, but the neurotransmitter used by these cells has not been determined. In the present study, we examine whether γ‐aminobutyric acid (GABA) or glycine could be the inhibitory neurotransmitter of BÖTZ cells. In chloralose‐anesthetized rats, we individually filled 20 physiologically characterized BÖTZ neurons with biotinamide by using a juxtacellular labeling method. Medullary sections containing the labeled BÖTZ neurons were processed for in situ hybridization by using digoxigenin‐labeled riboprobes for glutamic acid decarboxylase isoform 67 (GAD67), a marker for GABAergic neurons, or for glycine transporter 2 (GLYT2), a marker for glycinergic neurons. All BÖTZ cells examined contained GLYT2 mRNA (n = 10), whereas none had detectable levels of GAD67 mRNA (n = 10). For a positive control, 12 GABAergic neurons in the substantia nigra pars reticulata also were recorded and filled with biotinamide in vivo. Most of these cells, as expected, had detectable levels of GAD67 mRNA (11 out of 12). These results demonstrate that the juxtacellular labeling method can be combined with in situ hybridization to identify physiologically characterized cells with probable GABAergic or glycinergic phenotypes. Furthermore, these data suggest that BÖTZ neurons use the neurotransmitter glycine and not GABA to provide widespread inhibition of respiratory‐related neurons. J. Comp. Neurol. 407:583–597, 1999.

Preproenkephalin mRNA is expressed by C1 and non-C1 barosensitive bulbospinal neurons in the rostral ventrolateral medulla of the rat

The autonomic regions of the thoracolumbar spinal cord receive a dense enkephalinergic (ENK) innervation from supraspinal sources, including the rostral ventrolateral medulla (RVLM). In the present study, we sought to determine whether the barosensitive bulbospinal (BSBS) neurons of the RVLM express preproenkephalin (PPE) mRNA. After injection of Fluoro‐Gold (FG) into the upper thoracic spinal cord, neurons with PPE mRNA (PPE+ neurons) were retrogradely labeled throughout the ventrolateral medulla. At the most rostral RVLM level, 29% of bulbospinal PPE+ cells were tyrosine hydroxylase–immunoreactive (TH‐ir) and the latter constituted 19.4% of the bulbospinal TH‐ir cells. We determined whether the bulbospinal PPE+ RVLM neurons are barosensitive in two ways. First, we examined Fos production by FG‐labeled RVLM neurons after 2 hours of hydralazine‐induced hypotension (to 73 ± 2 mm Hg) in conscious rats. Hydralazine (10 mg/kg i.v.) increased the number of Fos‐ir neurons by two‐ to eightfold at all levels of the ventrolateral medulla examined. In the RVLM, 54% of bulbospinal PPE+ neurons were Fos‐ir, whereas such cells were more rarely found at caudal ventrolateral medullary levels. Second, we recorded individual BSBS RVLM units extracellularly in anesthetized rats and filled them juxtacellularly with biotinamide. Most biotinamide‐filled neurons were PPE+ (10 of 17), and the PPE+ BSBS cells had a faster axonal conduction velocity than those without PPE mRNA (4.2 vs. 0.67 m/sec). Four of the 10 PPE+ BSBS RVLM neurons were TH‐ir. In summary, PPE mRNA is predominantly expressed by RVLM BSBS neurons with lightly myelinated spinal axons. PPE mRNA is present in most noncatecholaminergic BSBS neurons and also in approximately 20% of the bulbospinal C1 neurons. BSBS RVLM neurons most likely provide a major ENK input to sympathetic preganglionic neurons and PPE mRNA is the first identified positive phenotype of the non‐C1 BSBS RVLM neurons. J. Comp. Neurol. 435:111–126, 2001.

Central respiratory modulation of barosensitive neurones in rat caudal ventrolateral medulla

The sympathetic nerves that maintain blood pressure are modulated by the central respiratory generator. Neurones in the rostral ventrolateral medulla (RVLM) that drive this sympathetic nerve activity (SNA) also display central respiratory drive (CRD)‐related activity, suggesting integration of respiratory and cardiovascular regulatory systems within the brainstem. Whether CRD‐related activity in the RVLM is due to direct inputs from central respiratory neurones or modulation of cardiovascular‐related neurones that influence the RVLM is not known. The caudal ventrolateral medulla (CVLM) contains GABAergic neurones that tonically inhibit presympathetic RVLM neurones and are essential for the production of numerous cardiovascular reflexes. The present study sought to determine whether cardiovascular‐related GABAergic neurones in the CVLM display CRD‐related activity. The firing patterns of individual barosensitive CVLM neurones were examined in relation to phrenic nerve activity in chloralose‐anaesthetized, ventilated, neuromuscularly blocked, vagotomized rats. Histograms of phrenic‐triggered CVLM neuronal activity showed that all baro‐activated CVLM neurones displayed one of four patterns of CRD‐related activity: (i) inspiratory peak (n= 15), (ii) inspiratory depression (n= 15), (iii) inspiratory peak with postinspiratory depression (n= 10), and (iv) postinspiratory peak (n= 9). A subset of each type of CVLM neurone was identified as GABAergic by individually filling the recorded neurone with biotinamide and observing expression of GAD67 mRNA by in situ hybridization (n= 10). These data suggest that the activity of GABAergic neurones in the CVLM is regulated by cardiovascular and respiratory inputs, and baro‐activated GABAergic CVLM neurones may contribute to CRD‐related modulation of presympathetic RVLM neurones and SNA.

VGLUT1 and VGLUT2 innervation in autonomic regions of intact and transected rat spinal cord

Fast excitatory neurotransmission to sympathetic and parasympathetic preganglionic neurons (SPN and PPN) is glutamatergic. To characterize this innervation in spinal autonomic regions, we localized immunoreactivity for vesicular glutamate transporters (VGLUTs) 1 and 2 in intact cords and after upper thoracic complete transections. Preganglionic neurons were retrogradely labeled by intraperitoneal Fluoro‐Gold or with cholera toxin B (CTB) from superior cervical, celiac, or major pelvic ganglia or adrenal medulla. Glutamatergic somata were localized with in situ hybridization for VGLUT mRNA. In intact cords, all autonomic areas contained abundant VGLUT2‐immunoreactive axons and synapses. CTB‐immunoreactive SPN and PPN received many close appositions from VGLUT2‐immunoreactive axons. VGLUT2‐immunoreactive synapses occurred on Fluoro‐Gold‐labeled SPN. Somata with VGLUT2 mRNA occurred throughout the spinal gray matter. VGLUT2 immunoreactivity was not noticeably affected caudal to a transection. In contrast, in intact cords, VGLUT1‐immunoreactive axons were sparse in the intermediolateral cell column (IML) and lumbosacral parasympathetic nucleus but moderately dense above the central canal. VGLUT1‐immunoreactive close appositions were rare on SPN in the IML and the central autonomic area and on PPN. Transection reduced the density of VGLUT1‐immunoreactive axons in sympathetic subnuclei but increased their density in the parasympathetic nucleus. Neuronal cell bodies with VGLUT1 mRNA occurred only in Clarkes column. These data indicate that SPN and PPN are densely innervated by VGLUT2‐immunoreactive axons, some of which arise from spinal neurons. In contrast, the VGLUT1‐immunoreactive innervation of spinal preganglionic neurons is sparse, and some may arise from supraspinal sources. Increased VGLUT1 immunoreactivity after transection may correlate with increased glutamatergic transmission to PPN. J. Comp. Neurol. 503:741–767, 2007.

Attenuated baroreflex control of sympathetic nerve activity in obese Zucker rats by central mechanisms.

Adult obese Zucker rats (OZRs) have reduced sympathetic responses to evoked changes in arterial pressure (AP) compared to lean Zucker rats (LZRs). This study examined whether attenuated sympathetic baroreflexes in OZRs may be due to altered sensory or central mechanisms. The OZRs had elevated baseline splanchnic sympathetic nerve activity (SNA) and mean AP (MAP) compared to age‐matched LZRs under urethane anaesthesia (P < 0.05). Aortic depressor nerve activity (ADNA) was measured while AP was altered by infusions of phenylephrine or nitroprusside (±60 mmHg over 60–90 s) in rats treated with atropine and propranolol to eliminate changes in heart rate. Although baseline ADNA was higher in the hypertensive OZRs, the relationship between MAP and ADNA was comparable in OZRs and LZRs. In contrast, electrical stimulation of the ADN afferent fibres (5 s train, 2 ms pulses, 4 V, 0.5–48 Hz) produced dramatically smaller reductions in SNA and MAP in OZRs compared to LZRs (P < 0.05). After blockade of α‐adrenergic receptors to prevent sympathetically mediated depressor responses, OZRs still had reduced sympathetic responses to stimulation of the ADN. In addition, stimulation of vagal afferent nerves electrically or with phenylbiguanide (1, 2, 4 and 8 μg, i.v.) produced smaller inhibitions of SNA in OZRs compared with LZRs (P < 0.05). These data suggest that attenuated sympathetic baroreflexes are the result of altered central mechanisms in OZRs, and not deficits in the responsiveness of aortic baroreceptors to AP. Furthermore, central deficits in the regulation of SNA in OZRs extend to other sympathoinhibitory reflexes initiated by vagal afferent nerves.

Altered sympathetic reflexes and vascular reactivity in rats after exposure to chronic intermittent hypoxia

Non‐technical summary  Exposure to chronic intermittent hypoxia (CIH) leads to chronically elevated sympathetic nerve activity (SNA) and mean arterial pressure (MAP) with exaggerated rises in SNA and MAP to activation of peripheral chemoreceptors. We show that CIH leads to augmented rises in SNA by stimulation of reflexes from other sensory nerves, suggesting a global change in how the brain processes sensory information. Furthermore, the ability of glutamate in the rostral ventrolateral medulla to increase SNA and MAP is enhanced after CIH, providing a potential brain mechanism for CIH‐induced exaggeration of sympathetic reflexes. Paradoxically, exaggerated sympathetic reflexes were accompanied by normal rises in MAP. In agreement, activation of adrenergic vascular receptors yields blunted rises in MAP in rats after CIH. These data suggest that exposure to CIH facilitates rises in SNA, potentially by changes in the brainstem, which are buffered by a reduction in the ability of sympathetic nerves to raise MAP.