Jaspreet K. Bassi

Leptin mediates the increase in blood pressure associated with obesity

Summary Obesity is associated with increased blood pressure (BP), which in turn increases the risk of cardiovascular diseases. We found that the increase in leptin levels seen in diet-induced obesity (DIO) drives an increase in BP in rodents, an effect that was not seen in animals deficient in leptin or leptin receptors (LepR). Furthermore, humans with loss-of-function mutations in leptin and the LepR have low BP despite severe obesity. Leptin’s effects on BP are mediated by neuronal circuits in the dorsomedial hypothalamus (DMH), as blocking leptin with a specific antibody, antagonist, or inhibition of the activity of LepR-expressing neurons in the DMH caused a rapid reduction of BP in DIO mice, independent of changes in weight. Re-expression of LepRs in the DMH of DIO LepR-deficient mice caused an increase in BP. These studies demonstrate that leptin couples changes in weight to changes in BP in mammalian species.

Expression of angiotensin type 1A receptors in C1 neurons restores the sympathoexcitation to angiotensin in the rostral ventrolateral medulla of angiotensin type 1A knockout mice.

In adult mice we determined whether expression of angiotensin II (Ang II) type 1A receptors (AT1ARs) in C1 neurons mediates the excitation of the rostral ventrolateral medulla (RVLM) by Ang II. Blood pressure, heart rate, and sympathetic nerve activity were measured in anesthetized, artificially ventilated wild-type (n=15) and AT1AR knockout (AT1A−/−; n=9) mice. Microinjection of Ang II (50 nL of 0.1 to 1.0 mmol/L) into the RVLM induced a dose-related, sympathetically mediated pressor response (maximum of 17±2 mm Hg) in wild-type mice. These microinjections had no effect in AT1A−/− mice. Endogenous AT1Rs occur on catecholaminergic C1 neurons in the RVLM. We induced AT1AR or green fluorescent protein expression in C1 neurons of AT1A−/− mice through bilateral microinjection of replication-deficient lentiviruses, with transgene expression under the control of a phox2 transcription factor binding promoter (PRSx8) (Lv-PRSx8-AT1A, n=10, and Lv-PRSx8-GFP, n=5). Transgene expression was observed in a significant proportion of RVLM C1 neurons. In anesthetized Lv-PRSx8-AT1A injected mice, unilateral RVLM microinjection of Ang II (50 nL of 1 mmol/L) increased blood pressure (17±4 mm Hg) and sympathetic nerve activity (155±32%). No response to Ang II occurred in Lv-PRSx8-GFP microinjected mice. These results show that Ang II–mediated excitation of RVLM neurons in adult mice depends on the AT1AR with little or no effect of type 1B or 2 receptors. Expression of the AT1AR predominantly in C1 catecholamine neurons restores the response to Ang II in the AT1A−/− mouse and demonstrates that these neurons are sympathoexcitatory in the mouse.

Angiotensin Type 1A Receptors in C1 Neurons of the Rostral Ventrolateral Medulla Modulate the Pressor Response to Aversive Stress

The rise in blood pressure during an acute aversive stress has been suggested to involve activation of angiotensin type 1A receptors (AT1ARs) at various sites within the brain, including the rostral ventrolateral medulla. In this study we examine the involvement of AT1ARs associated with a subclass of sympathetic premotor neurons of the rostral ventrolateral medulla, the C1 neurons. The distribution of putative AT1AR-expressing cells was mapped throughout the brains of three transgenic mice with a bacterial artificial chromosome-expressing green fluorescent protein under the control of the AT1AR promoter. The overall distribution correlated with that of the AT1ARs mapped by other methods and demonstrated that the majority of C1 neurons express the AT1AR. Cre-recombinase expression in C1 neurons of AT1AR-floxed mice enabled demonstration that the pressor response to microinjection of angiotensin II into the rostral ventrolateral medulla is dependent upon expression of the AT1AR in these neurons. Lentiviral-induced expression of wild-type AT1ARs in C1 neurons of global AT1AR knock-out mice, implanted with radiotelemeter devices for recording blood pressure, modulated the pressor response to aversive stress. During prolonged cage-switch stress, expression of AT1ARs in C1 neurons induced a greater sustained pressor response when compared to the control viral-injected group (22 ± 4 mmHg for AT1AR vs 10 ± 1 mmHg for GFP; p < 0.001), which was restored toward that of the wild-type group (28 ± 2 mmHg). This study demonstrates that AT1AR expression by C1 neurons is essential for the pressor response to angiotensin II and that this pathway plays an important role in the pressor response to aversive stress.

Central Neural Regulation of Cardiovascular Function by Angiotensin: A Focus on the Rostral Ventrolateral Medulla

Angiotensin II acts through specific receptors to alter several brain functions including fluid and electrolyte control, neuroendocrine function and autonomic efferent activity. This review discusses one brain site, the rostral ventrolateral medulla, where the actions of angiotensin II have been intensively studied. The rostral ventrolateral medulla plays a critical role in the generation and regulation of sympathetic activity to the cardiovascular system and hence is important for blood pressure control. Current evidence indicates that angiotensin II activates neurons in the rostral ventrolateral medulla via the AT1A receptor. In some models of cardiovascular disease, blockade of AT1 receptors in the rostral ventrolateral medulla reduces sympathetic nerve activity and blood pressure suggesting that overactivity of the angiotensin system in this nucleus may play a role in the maintenance of high blood pressure.

Regulation of angiotensinogen by angiotensin II in mouse primary astrocyte cultures

J. Neurochem. (2011) 119, 18–26.

Efferent projections of C3 adrenergic neurons in the rat central nervous system

C3 neurons constitute one of three known adrenergic nuclei in the rat central nervous system (CNS). While the adrenergic C1 cell group has been extensively characterized both physiologically and anatomically, the C3 nucleus has remained relatively obscure. This study employed a lentiviral tracing technique that expresses green fluorescent protein behind a promoter selective to noradrenergic and adrenergic neurons. Microinjection of this virus into the C3 nucleus enabled the selective tracing of C3 efferents throughout the rat CNS, thus revealing the anatomical framework of C3 projections. C3 terminal fields were observed in over 40 different CNS nuclei, spanning all levels of the spinal cord, as well as various medullary, mesencephalic, hypothalamic, thalamic, and telencephalic nuclei. The highest densities of C3 axon varicosities were observed in Lamina X and the intermediolateral cell column of the thoracic spinal cord, as well as the dorsomedial medulla (both commissural and medial nuclei of the solitary tract, area postrema, and the dorsal motor nucleus of the vagus), ventrolateral periaqueductal gray, dorsal parabrachial nucleus, periventricular and rhomboid nuclei of the thalamus, and paraventricular and periventricular nuclei of the hypothalamus. In addition, moderate and sparse projections were observed in many catecholaminergic and serotonergic nuclei, as well as the area anterior and ventral to the third ventricle, Lamina X of the cervical, lumbar, and sacral spinal cord, and various hypothalamic and telencephalic nuclei. The anatomical map of C3 projections detailed in this survey hopes to lay the first steps toward developing a functional framework for this nucleus. J. Comp. Neurol. 520:2352–2368, 2012.

C1 neurons in the rat rostral ventrolateral medulla differentially express vesicular monoamine transporter 2 in soma and axonal compartments

Vesicular monoamine transporter 2 (VMAT2) packages biogenic amines into large dense core and synaptic vesicles for either somatodendritic or synaptic release from neurons of the CNS. Whilst the distribution of VMAT2 has been well characterized in many catecholaminergic cell groups, its localization amongst C1 adrenergic neurons in the medulla has not been examined in detail. Within the rostral ventrolateral medulla (RVLM), C1 neurons are a group of barosensitive, adrenergic neurons. Rostral C1 cells project to the thoracic spinal cord and are considered sympathetic premotor neurons. The majority of caudal C1 cells project rostrally to regions such as the hypothalamus. The present study sought to quantitate the somatodendritic expression of VMAT2 in C1 neurons, and to assess the subcellular distribution of the transporter. Immunoreactivity for VMAT2 occurred in 31% of C1 soma, with a high proportion of these in the caudal part of the RVLM. Retrograde tracing studies revealed that only two of 43 bulbospinal C1 neurons contained faint VMAT2‐immunoreactivity, whilst 88 ± 5% of rostrally projecting neurons were VMAT2‐positive. A lentivirus, designed to express green fluorescent protein exclusively in noradrenergic and adrenergic neurons, was injected into the RVLM to label C1 neurons. Eighty‐three percent of C1 efferents that occurred in close proximity to sympathetic preganglionic neurons within the T3 intermediolateral cell column contained VMAT2‐immunoreactivity. These data demonstrate differential distribution of VMAT2 within different subpopulations of C1 neurons and suggest that this might reflect differences in somatodendritic vs. synaptic release of catecholamines.

Distribution of cells expressing human renin-promoter activity in the brain of a transgenic mouse

Renin plays a critical role in fluid and electrolyte homeostasis by cleaving angiotensinogen to produce Ang peptides. Whilst it has been demonstrated that renin mRNA is expressed in the brain, the distribution of cells responsible for this expression remains uncertain. We have used a transgenic mouse approach in an attempt to address this question. A transgenic mouse, in which a 12.2 kb fragment of the human renin promoter was used to drive expression of Cre-recombinase, was crossed with the ROSA26-lac Z reporter mouse strain. Cre-recombinase mediated excision of the floxed stop cassette resulted in expression of the reporter protein, beta-galactosidase. This study describes the distribution of beta-galactosidase in the brain of the crossed transgenic mouse. In all cases where it was examined the reporter protein was co-localized with the neuronal marker NeuN. An extensive distribution was observed with numerous cells labeled in the somatosensory, insular, piriform and retrosplenial cortices. The motor cortex was devoid of labeled cells. Several other regions were labeled including the parts of the amygdala, periaqueductal gray, lateral parabrachial nucleus and deep cerebellar nuclei. Overall the distribution shows little overlap with those regions that are known to express receptors for the renin-angiotensin system in the adult brain. This transgenic approach, which demonstrates the distribution of cells which have activated the human renin promoter at any time throughout development, yields a unique and extensive distribution of putative renin-expressing neurons. Our observations suggest that renin may have broader actions in the brain and may indicate a potential for interaction with the (pro)renin receptor or production of a ligand for non-AT(1)/AT(2) receptors.

Excessive Respiratory Modulation of Blood Pressure Triggers Hypertension

The etiology of hypertension, the worlds biggest killer, remains poorly understood, with treatments targeting the established symptom, not the cause. The development of hypertension involves increased sympathetic nerve activity that, in experimental hypertension, may be driven by excessive respiratory modulation. Using selective viral and cell lesion techniques, we identify adrenergic C1 neurons in the medulla oblongata as critical for respiratory-sympathetic entrainment and the development of experimental hypertension. We also show that a cohort of young, normotensive humans, selected for an exaggerated blood pressure response to exercise and thus increased hypertension risk, has enhanced respiratory-related blood pressure fluctuations. These studies pinpoint a specific neuronal target for ameliorating excessive sympathetic activity during the developmental phase of hypertension and identify a group of pre-hypertensive subjects that would benefit from targeting these cells.

Stimulation of Angiotensin Type 1A Receptors on Catecholaminergic Cells Contributes to Angiotensin-Dependent Hypertension

Hypertension contributes to multiple forms of cardiovascular disease and thus morbidity and mortality. The mechanisms inducing hypertension remain unclear although the involvement of homeostatic systems, such as the renin–angiotensin and sympathetic nervous systems, is established. A pivotal role of the angiotensin type 1 receptor in the proximal tubule of the kidney for the development of experimental hypertension is established. Yet, other systems are involved. This study tests whether the expression of angiotensin type 1A receptors in catecholaminergic cells contributes to hypertension development. Using a Cre-lox approach, we deleted the angiotensin type 1A receptor from all catecholaminergic cells. This deletion did not alter basal metabolism or blood pressure but delayed the onset of angiotensin-dependent hypertension and reduced the maximal response. Cardiac hypertrophy was also reduced. The knockout mice showed attenuated activation of the sympathetic nervous system during angiotensin II infusion as measured by spectral analysis of the blood pressure. Increased reactive oxygen species production was observed in forebrain regions, including the subfornical organ, of the knockout mouse but was markedly reduced in the rostral ventrolateral medulla. These studies demonstrate that stimulation of the angiotensin type 1A receptor on catecholaminergic cells is required for the full development of angiotensin-dependent hypertension and support an important role for the sympathetic nervous system in this model.