Physical (In)Activity-Dependent Structural Plasticity in Bulbospinal Catecholaminergic Neurons of Rat Rostral Ventrolateral Medulla 

The RVLM contains neurons that are integral to control of sympathetic nerve activity and blood pressure. These neurons are "connected" to the spinal cord and they play a part in regulating blood pressure throughout the body. Previous studies have shown that cardiovascular responses to the activation of RVLM neurons are increased in animals that are physically inactive, in comparison to physically active animals. Studies also show that when compared with physically active rats, sedentary rats show enhanced splanchnic sympathetic nerve responses. Considering these findings, the researchers hypothesized that, "Changes in the function of RVLM neurons that regulate splanchnic sympathetic outflow contribute to the development of hypertension". It has also been found that wheel running exercise affects the dendritic morphology of neurons in cardiorespiratory centers within the central nervous system. Based on this knowledge, it was hypothesized that, "Physical inactivity may increase sympathetic nerve activity by altering the structure of bulbospinal neurons in the RVLM, specifically through an increase in dendritic branching and/or an increase in the size of their cell bodies." 

To test these hypotheses the researchers observed two groups of rats, one sedentary group, and one physically active group, as well as one rat that was part of neither group . The sedentary group had no way to exercise, while the active group had a running wheel in their cage. The rats were observed for 11-12 weeks and each day running data was recorded from the active rats. After 11 or 12 weeks, depending on the rat, a retrograde tracer was injected into their spinal cord and then they were returned to their cages. One week after the injection of the tracer, all rats were perfused, brains and spinal cords were removed and postfixed for 3-4 days. Brainstems were stored briefly and then they were sectioned off and each section was distributed into a well. The sections of brainstems were distributed so that each well contained one section from a sedentary rat, and one section from a physically active rat. The brainstem of the rat that was not a part of either group was divided as well. 

The brainstem sections were then immunohistochemically stained, and embedded in resin. To detect the retrograde tracer that had previously been injected into the rat spinal cords, sections were treated with rabbit and goat antibodies. Next, the sections from the six active and five inactive rats were embedded in Durcupan resin on glass slides. The slides were then examined and photographed using a microscope equipped with a camera. Neurons were examined and counted in each brainstem section that showed the retrograde tracer, however, only neurons within a specified area of each section were counted.

After examination of these neurons, it was found that on average, neurons from sedentary versus active rates had more dendritic branch points, greater total dendrite length, greater total dendritic surface area, and more intersections in Sholl analyses. There was no difference between sedentary and active rats in cell body perimeter, cell body area, cell body shape, or number of primary dendrites. Furthermore, it was found that neurons located rostral to the caudal pole of FN had more branch points than those located caudal to the caudal pole of FN, in sedentary rats, while the morphology of neurons in physically active rats remained consistent throughout the RVLM. This finding supports the hypothesis that physical activity may alter the structure of bulbospinal neurons in RVLM. It was also found that when compared with physically active rats, sedentary rats show increased baseline renal sympathetic nerve activity. The sympathetic outflow of sedentary rats was found to be nearly twice as much as that of active rats in splanchnic sympathetic nerve activity. In addition, neurons within the RVLM show increased activation in various disorders, such as high-salt diet, hyperinsulinemia, chronic renal failure, obesity, hypertension, and chronic heart failure. These findings suggest that neurons in the RVLM are directly involved in the regulation of blood pressure within the body. 

PVH and Neuron Projections

It is widely accepted that the hypothalamus has neurons that are responsible for the regulation of arterial pressure. The PVH has known projects to the RVLM, which is a major brain stem region responsible for the regulation of sympathetic nerve activity. Furthermore, the PVH also has neuronal projections to the NTS which is paramount is receiving afferent inputs from the cardiovascular system and relaying vasomotor effects on the PVH and ultimately the RVLM. The NTS also has projections to presympathetic ganglionic neurons within the spinal cord that influence SNA and regulation cardiovascular function. The RVLM and NTS have been shown to have separate hypothalamic inputs however; no attempts have shown hypothalamic inputs to both structures. In this experiment by Badoer, they attempted to identify hypothalamic neurons having projections to functionally identified areas of the RVLM and NTS. Male Sprague Dawley rats were anesthetized and injected with tracers DY (diamidino yellow) and FB (fast blue). The animals were allowed to recover for 3-5 days for transport. Animals were sacrificed and the brains were sliced and studied using fluorescence microscopy. The results showed that numerus labeled neurons were shown to have projections to along the rostral-caudal axis of the hypothalamus. Most interestingly, the neurons in the hypothalamus had project to the RVLM and NTS were overlapping in the hypothalamus. Despite this finding, Badoer concludes that double labeled neurons were rare which would indicate that neurons from the hypothalamus to the RVLM and NTS have direct projections and influence to one or the other, not both. It does seem likely thought that both overlapping neurons that project to the RVLM or NTS may influence each other through synaptic contacts. Badoer also located neurons with the PVH that project to the NTS are found more ventral in the hypothalamus whereas projections to the RVLM were found more throughout the hypothalamus. Overall, this experiment by Badoer is critical in understanding neuronal projections to and from the hypothalamus to keep regions responsible for cardiovascular function.

Brief PVH review

The PVN is anatomically composed of two types of neurons, magnocellular and parvocellular neurons. Both types of neurons are further subdivided into three magnocelluar and five parvocellular neurons. The magnocellular subdivisions are known as anterior, posterior, and medial subnuclei that project to the neurohypophysis and are responsible for the production of posterior pituitary hormones. The parvocellular neurons are subdivided into dorsal, lateral, medial, periventricular, and anterior subnuclei. These regions project to the autonomic nuclei in the brain stem as well as the spinal cord and are responsible for cardiovascular regulation through activation of sympathetic nervous system. The major regulator of the sympathetic nervous system is the RVLM which has numerous projections from the PVN that influence its regulation of arterial pressure.

Information regarding cardiovascular regulation reaches the PVH through a hindlimb brain region known as the NTS. The NTS is the main site of terminating fibers from various cardiovascular receptors such as the baroreceptors, chemoreceptors, and cardiopulmonary receptors. Axons from the caudal portion of the NTS have been found to terminate in the parvocellular and dorsal cap regions of the PVH however, the final target is not known yet.

PVH neurons are continuously active and subject to tonic inhibition arising from GABA and nitric oxide. Administration of NO causes as a decrease in sympathetic nerve activity and it has been found that the majority of the NO is the magnocellular neurons and it is hypothesized that magnocellular neurons may contribute to the autonomic regulation of SNA.  A functional experiment was performed which reported that administration of sodium nitroprusside into the PVH decreased rSNA, AP, and heart rate. Furthermore, administration of NO antagonist blocked the inhibitory effect of the NO on the SNA indicating that NO is inhibitory to sympathetic outflow.

Overall, the paper reviews several ways to examine the PVH and concludes that regulating synaptic activity of the PVH at the level of the parvocellular neurons contributes to sympathetic control and setting basal activity levels. In setting this basal tone, NO, GABA, glutamate and vasopressin are all contributors to tonic activity of the PVH. Therefore, disturbances in these pathways can lead to various cardiovascular disease states.

MEMRI and Hypothalamic in vivo measurements

The paper submitted to NMR in Biomedicine by Yu-Ting Kuo et al 2006, used MEMRI to detect hypothalamic neuronal activity in mice in fasting and non-fasting states. MEMRI is an in vivo technique that uses Mn²⁺ as a Ca²⁺ surrogate to estimate neuronal activity. Mn²⁺ enters excitable cells it has been proven to be a viable contrast agent for MRI causing shortened T₁ relaxation times. The paper focused on the paraventricular nucleus of the hypothalamus is an important brain region responsible for regulation of sympathetic nervous system, hormone secretion, homeostasis and appetite. The current study examined differences in the neuronal activity between fasting and non-fasting states in mice between 16-24 weeks old. Administration of MnCl₂ occurred through the tail vein following implantation of a cannula. A control group of animals had access to ad libitum (n=4) while non-fasting animals had food removed 12-16 hours prior to scanning. All scans began at 9am with three baseline scans before following by continuous slow infusion of MnCl­₂ and sixty-three scans were performed over the course of 2 hour with an average individual scan time of 1 minutes 57 seconds. The results showed approximately 20% signal intensity increase from baseline scans in the PVH, arcuate hypothalamus (Arc), VMH, AP, and fourth ventricle. Each image was normalized to saline phatoms (SI tissue/saline phantom). Overnight fasting lead to significant increases in enhancement in PVH and VHM compared to non-fasted animals (p=0.04). The studies finding indicate that higher neuronal activity is found it the PVN of fasted animals compared to non-fasted animals. Finally, MEMRI is able to determine differences in enhancement in between feeding states and can further our understanding of the PVH based on its various regulatory functions.

PVH and heart failure

The topic of this paper examines cellular and molecular mechanisms in the PVH in the pathophysiological state of heart failure. A key aspect of heart failure is the inability to regulate the sympathetic nervous system. The PVH is a key regulatory brain region responsible for sympathetic regulation and ultimately arterial pressure. Afferent projections from the cardiovascular system to the NTS initiate cardiovascular regulation through projects to the PVH and the RVLM. The PVH and RVLM subsequently adjust their sympathetic response from the NTS to regulate homeostasis. Furthermore, the PVH sends efferent projections to the sympathetic preganglionic neurons of the RVLM and IML to regulate sympathetic tone. The IML is critical in the maintenance of arterial pressure since it regulates sympathetic preganglionic neurons of the entire body.

The PVH alone is an important regulator of arterial pressure. The three important subsets of neurons within the PVH are the magnocellular, the parvocellular neuroendocrine, and parvocellular pre-autonomic neurons. The magnocellular neurons synthesize and secrete vasopressin and oxytocin which are released into circulation through the posterior pituitary. The parvocellular neuroendocrine hormones release GHRH to the anterior pituitary via the hypophyseal portal vessels. Finally, the parvocellular pre autonomic neurons regulate sympathetic nerve activity. An important aspect of the PVH is that the parvocellular pre autonomic neurons project reciprocally to the NTS as well as the RVLM and IML to regulate arterial pressure.

 A critical discovery by Pyner, demonstrated at least 4 pathways from the NTS project target PVH neurons: spinally projecting neurons, nNOS-magnocellular neurons, GABA interneurons, and nNOS containing interneurons bordering the PVH. This discovering is important in being able to assess sensory inputs to the NTS with the PVH.

The PVH is under tonic inhibitory control via NO mediated GABA inhibition. Excitation of the PVH requires NMDA mediated glutamate driven activation in the PVN. A major finding implicated the NDMA glutamate stimulation in heart failure since no increases in blood pressure were discovered during tonic glutamate administration. However, when sympathetic nerve activity is enhanced in conditions of heart failure, administration of a NMDA antagonist decreased sympathetic nerve activity. This raised the question of why and how this happens if tonic increases in glutamate did not give rise to sympathetic nerve activity in normal conditions. Increasing evidence suggests angiontensin 2 and downregulation of nNOS an enzyme responsible for NO generation.

The paper elaborates on the PVH and finds new projections to the PVH from the NTS as well as examines the effects that heart failure has on PVH. However, information has not been discovered yet to determine a viable drug target for regulating many of these pathways in heart failure.