An immunohistochemical investigation of the relationship between neuronal nitric oxide synthase, GABA and presympathetic paraventricular neurons in the hypothalamus

doi:10.1016/j.neuroscience.2009.01.012

Neuroscience

Volume 159, Issue 3, 31 March 2009, Pages 1079-1088

https://doi.org/10.1016/j.neuroscience.2009.01.012 Get rights and content

Abstract

Functional studies suggest that nitric oxide (NO) modulates sympathetic outflow by enhancing synaptic GABAergic function. Furthermore, the paraventricular nucleus of the hypothalamus (PVN), an important site for autonomic and endocrine homeostasis constitutes an important center mediating NO actions on sympathetic outflow. However, the exact anatomical organization of GABA and NO releasing neurons with the PVN neurons that regulate autonomic activity is poorly understood. The present study addressed this by identifying PVN-presympathetic neurons in the rat with the retrograde tracer Fluorogold injected into T2 segment of the spinal cord or herpes simplex virus injected into the adrenal medulla (AM). GABAergic or nitric oxide cell bodies were identified by antibodies directed towards GABA or glutamate decarboxylase (GAD67) enzyme or neuronal nitric oxide synthase. This revealed a population of GABAergic neurons to be synaptically associated with a chain of pre-sympathetic neurons targeting the AM. Furthermore, this GABAergic population is not a cellular source of NO. Within the PVN, the majority of cellular nitric oxide was localized to non-spinally projecting neurons while for the PVN-spinally projecting neuronal pool only a minority of neuron were immunopositive for neuronal nitric oxide synthase. In summary, nitrergic and GABAergic neurons are associated with a hierarchical chain of neurons that regulate autonomic outflow. This anatomical arrangement supports the known function role of a NO-GABA modulation of sympathetic outflow.

Section snippets

Experimental procedures

All experiments were approved by the local ethics committee of Durham University and performed under a Home Licence in accordance with the UK Animal (Scientific Procedures) Act 1986. Furthermore, all surgical procedures were carried out on anesthetized animals that minimized suffering with the minimum number of animals used. Animals were killed with an overdose of Do-lethal (sodium pentobarbital 60 mg/kg, Vetquinol, Bucks, UK) at the termination of the experiment.

Distribution of HSV-GFP neurons within the PVN

Injection of HSV-GFP into the AM labeled a target specified chain of presympathetic neurons that include PVN-spinally projecting neurons. HSV-GFP was localized to the nucleus of the neurons and labeled neurons were mainly confined to the dorsal cap, medial and lateral parvocellular regions of the PVN (Figs. 1A, E and 2E). Furthermore, the HSV-GFP neurons spread dorsally and laterally beyond the boundaries of the PVN nucleus. The number of AM projecting neurons was 370±69 (SEM; n=9).

Relationship between HSV-GFP containing and nNOS-IR neurons

Neuronal

Discussion

This study provides new findings revealing important details of the neural circuitry within the PVN that is associated with a chain of pre-sympathetic neurons projecting to the AM. Labeling only spinally projecting neurons and comparing with transynaptic labeling of the chain of neurons, it is shown that GABA neurons within the PVN are closely associated with spinally projecting neurons, a few of which were NOS positive. Previous studies have demonstrated that within the PVN there is a tonic

Conclusion

Using GABA and nNOS immunohistochemistry combined with retrograde tracing has revealed GABAergic and nNOS synthesizing neurons to be part of the neural circuitry regulating AM sympathetic function. In addition, GABAergic neurons are not capable of synthesizing NO. As such a neuronal source of NO to modulate GABA function in the reflex control of sympathetic activity is derived from two populations localized to the PVN and surrounding area, the magnosecretory neurons and a presumed NOS

Acknowledgments

This work was supported by the British Heart Foundation. We thank Dr. A. Buchan and P. McLeish for technical assistance related to the HSV-GFP experiments and Professor J. H. Coote for helpful discussions in the preparation of the manuscript.

References (48)

R.E. Brooke et al.
Spinal cord interneurones labeled transneuronally from the adrenal gland by a GFP-herpes construct contain the potassium channel subunit Kv3.1b
Auton Neurosci Basic Clin
(2002)
J.P. Card
Practical considerations for the use of pseudorabies virus in transneuronal studies of neural circuitry
Neurosci Biobehav Rev
(1998)
Q. Chen et al.
Regulation of synaptic input to hypothalamic presympathetic neurons by GABA_B receptors
Neuroscience
(2006)
R.J. Dado et al.
Evidence that fluorogold can be transported avidly through fibers of passage
Brain Res
(1990)
V.S. Fénelon et al.
Cellular localization and differential distribution of GABA_A receptor subunit proteins and messenger RNAs within hypothalamic magnocellular neurons
Neuroscience
(1995)
A. Kantzides et al.
nNOS-containing neurons in the hypothalamus and medulla project to the RVLM
Brain Res
(2005)
Y. Li et al.
Nitric oxide inhibits the firing activity of hypothalamic paraventricular neurons that innervate the medulla oblongata: role of GABA
Neuroscience
(2003)
A.D. Loewy
Viruses as transneuronal tracers for defining neural circuits
Neurosci Biobehav Rev
(1998)
S. Pyner et al.
Identification of an efferent projection from the paraventricular nucleus of the hypothalamus terminating close to spinally projecting rostral ventrolateral medullary neurons
Neuroscience
(1999)
S. Pyner et al.
Identification of branching paraventricular neurons of the hypothalamus that project to the rostral ventrolateral medulla and spinal cord
Neuroscience
(2000)

C.B. Saper et al.

Direct hypothalamic autonomic connection

Brain Res

(1976)

L.C. Schmued et al.

Fluorogold: a new fluorescent retrograde axonal tracer with numerous unique properties

Brain Res

(1986)

B.R. Schofield et al.

On the use of retrograde tracers for identification of axon collateral with multiple fluorescent retrograde tracers

Neuroscience

(2007)

A.D. Shafton et al.

Neurons in the hypothalamic paraventricular nucleus send collaterals to the spinal cord and to the rostral ventrolateral medulla in the rat

Brain Res

(1998)

J.E. Stern

Nitric oxide and homeostatic control: an intracellular signaling molecule contributing to autonomic and neuroendocrine integration

Prog Biophys Mol Biol

(2004)

A.M. Strack et al.

CNS cell groups regulating the sympathetic outflow to adrenal gland as revealed by transneuronal cell body labeling with pseudorabies virus

Brain Res

(1989)

M.L. Weiss et al.

Neural circuitry of the kidney: NO-containing neurons

Brain Res

(2001)

Q. Yang et al.

Kv1.1/1.2 channels are downstream effectors of nitric oxide on synaptic GABA release to preautonomic neurons in the paraventricular nucleus

Neuroscience

(2007)

Z. Yang et al.

Paraventricular nucleus activation of renal sympathetic neurons is synaptically depressed by nitric oxide and glycine at a spinal level

Neuroscience

(2004)

C. Boudaba et al.

Physiological mapping of local inhibitory inputs to the hypothalamic paraventricular nucleus

J Neurosci

(1996)

J.H. Coote

A role for the paraventricular nucleus of the hypothalamus in the autonomic control of the heart and kidney

Exp Physiol

(2005)

C. Decavel et al.

GABA: a dominant neurotransmitter in the hypothalamus

J Comp Neurol

(1990)

L.W. Enquist et al.

Infection and spread of alphaherpes viruses in the nervous system

Adv Virus Res

(1999)

V.S. Fénelon et al.

Plasticity in GABA_A receptor subunit mRNA expression by hypothalamic magnocellular neurons in the adult rat

J Neurosci

(1996)

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Nitric oxide in the PVN is generated by the enzyme neuronal nitric oxide synthase with the resultant NO gas readily diffusing across cell membranes to activate cGMP mechanisms to exert it biological effects (Kennedy, 2000). Unlike in PVN parvocellular neurons, the NOS1 enzyme is widespread in the magnocellular population, nitrergic interneurons, and glial cells (Stern, 2004; Watkins et al., 2009; Stern and Filose, 2013) and the gas generated by these sources diffuses to the parvocellular neurons to modulate their activity (Stern, 2004; Stern and Filose, 2013). Administration of sodium nitroprusside (NO donor) into the PVN elicits decreases in RSNA while L-NAME (NO inhibitor) increases RSNA (Zhang et al., 1997).
Neuroanatomic and functional studies show the paraventricular (PVN) of the hypothalamus to have a central role in the autonomic control that supports cardiovascular regulation. Direct and indirect projections from the PVN preautonomic neurons to the sympathetic preganglionic neurons in the spinal cord modulate sympathetic activity. The preautonomic neurons of the PVN adjust their level of activation in response to afferent signals arising from peripheral viscerosensory receptors relayed through the nucleus tractus solitarius. The prevailing sympathetic tone is a balance between excitatory and inhibitory influences that arises from the preautonomic PVN neurons. Under physiologic conditions, tonic sympathetic inhibition driven by a nitric oxide—γ-aminobutyric acid-mediated mechanism is dominant, but in pathologic situation such as heart failure there is a switch from inhibition to sympathoexcitation driven by glutamate and angiotensin II. Angiotensin II, reactive oxygen species, and hypoxia as a result of myocardial infarction/ischemia alter the tightly regulated posttranslational protein–protein interaction of CAPON (carboxy-terminal postsynaptic density protein ligand of neuronal nitric oxide synthase (NOS1)) and PIN (protein inhibitor of NOS1) signaling mechanism. Within the preautonomic neurons of the PVN, the disruption of CAPON and PIN signaling leads to a downregulation of NOS1 expression and reduced NO bioavailability. These data support the notion that CAPON-PIN dysregulation of NO bioavailability is a major contributor to the pathogenesis of sympathoexcitation in heart failure.
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We did not determine the mechanism by which NO, acting within preautonomic PVN neurons, changes autonomic activity in trained rats and in sedentary ones supplemented with l-Arginine, but it is largely possible that NO alleviates peripheral sympathetic activity by improving local inhibitory gabaergic mechanism. Accumulated evidence in the literature has shown that NO enhances gabaergic synaptic activity, inhibits the firing of preautonomic PVN neurons innervating brainstem areas and reduces renal sympathetic nerve discharge [6–9,11] and that exercise training potentiates the gabaergic inhibitory mechanism within the PVN [49,50]. A further confirmation of NO-induced sympathetic inhibition was given by the effect of PVN N-Propyl microinjection on baroreflex control: it specifically abrogated training-induced and l-arginine-induced augmentation of the upper plateau, an effect mainly mediated by increased sympathetic outflow during transient pressure decreases.
Nitric oxide (NO), an intercellular signaling molecule is relevant for circulatory autonomic control. Brain NO synthase (NOS) and NO levels were downregulated in pathological conditions, but rescued after exercise training. We hypothesized that exercise training was also able to improve NO modulation within the hypothalamic paraventricular nucleus (PVN) of healthy rats. Male Wistar rats were submitted to two 4-weeks protocols: i) swimming training (T) or kept sedentary (S), ii) l-arginine (62,5 mg/mL, 1 mL/day p. o.) or vehicle supplementation. Rats underwent stereotaxic surgery (PVN bilateral guide cannulas) and chronic catheterization of artery/vein. Arterial pressure (AP), heart rate (HR) and baroreflex sensitivity were recorded in conscious rats at rest and following a selective nNOS inhibitor (Nw-Propyl-l-Arginine, 4 nmol/100 nL) within the PVN. Rats were deeply anesthetized for brain perfusion/harvesting after respiratory arrest. In separate groups (T and S, l-arginine and Vehicle supplemented) not submitted to PVN cannulation, fresh and fixed brains were obtained for gene and protein nNOS expression (qPCR and immunohistochemistry) and nitrite levels (Griess reaction). T and l-arginine treatment were accompanied by resting bradycardia, augmented parasympathetic and reduced sympathetic activity to heart and vessels (power spectral analysis) and increased baroreflex sensitivity (^†P < 0.05). In contrast, PVN nNOS inhibition blocked/attenuated these effects in addition to significantly increase in resting MAP and HR (with larger effects in T and l-arginine treated rats vs. respective controls, ^†P < 0.05). T increased nNOS gene and protein expression within the ventromedial and posterior PVN nuclei (^†P < 0.05). PVN nitirite levels were also increased in T and l-arginine groups (^†P < 0.05). Data strongly suggest that training by increasing NO availability within PVN preautonomic nuclei favors both the slow down of sympathetic and the augmentation of parasympathetic activity and facilitates baroreflex control, therefore improving autonomic regulation of the heart in healthy rats.
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NeuroanatomyAn immunohistochemical investigation of the relationship between neuronal nitric oxide synthase, GABA and presympathetic paraventricular neurons in the hypothalamus

Abstract

Section snippets

Experimental procedures

Distribution of HSV-GFP neurons within the PVN

Relationship between HSV-GFP containing and nNOS-IR neurons

Discussion

Conclusion

Acknowledgments

Auton Neurosci Basic Clin

Neurosci Biobehav Rev

Neuroscience

Brain Res

Neuroscience

Brain Res

Neuroscience

Neurosci Biobehav Rev

Neuroscience

Neuroscience

Brain Res

Brain Res

Neuroscience

Brain Res

Prog Biophys Mol Biol

Brain Res

Brain Res

Neuroscience

Neuroscience

Physiological mapping of local inhibitory inputs to the hypothalamic paraventricular nucleus

J Neurosci

A role for the paraventricular nucleus of the hypothalamus in the autonomic control of the heart and kidney

Exp Physiol

GABA: a dominant neurotransmitter in the hypothalamus

J Comp Neurol

Infection and spread of alphaherpes viruses in the nervous system

Adv Virus Res

Plasticity in GABAA receptor subunit mRNA expression by hypothalamic magnocellular neurons in the adult rat

J Neurosci

Neuroanatomy
An immunohistochemical investigation of the relationship between neuronal nitric oxide synthase, GABA and presympathetic paraventricular neurons in the hypothalamus

Plasticity in GABA_A receptor subunit mRNA expression by hypothalamic magnocellular neurons in the adult rat