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Cardiovascular
Neuroscience Laboratory
Research Projects
Chronic heart failure induces changes in autonomic nerve
pathways in the paraventricular nucleus of the hypothalamus
The
paraventricular nucleus of the hypothalamus (PVN) plays an important
role in cardiovascular control. It integrates information from
many visceral afferents and influences three major central processing
systems, the endocrine, neuroendocrine and autonomic nervous
systems. the interactions between these systems is unbalanced
in chronic heart failure (CHF), a major health problem in developed
nations that results from the heart’s inability to pump blood
effectively. The PVN compensates for the failing heart through
alterations in hormone release from the pituitary and in the activity
of central circuits that control sympathetic outflow, particularly
cardiac sympathetic outflow. These effects suggest that autonomic
nerve pathways in the PVN may be altered in CHF.
To address this
hypothesis, we performed left coronary ligation surgery in rats,
which is known to produce the symptoms of CHF in about 1/3 of surviving
rats after 6-7 weeks. Rats were perfused at that time and sections
of hypothalamus were stained immunohistochemically for corticotrophin
releasing factor (CRF), vasopressin (VP), nitric oxide synthase
(NOS), enkephalin (ENK) and pro-enkephalin (pro-ENK). Numbers of
immunoreactive neurons were counted in rats with CHF (i.e., rats
with infarcts occupying more than 40% of the left ventricle) and
in sham-operated control rats.
CRF-immunoreactive neurons were
more numerous in the PVN of rats with CHF than in control rats
(205 ± 108 vs 87 ± 16,
mean + SD; p<0.05, n=6 rats/group) as were VP-immunoreactive
neurons (289 ± 36 vs 232 ± 42; p<0.05, n=5 rats/group).
There was a trend towards a decrease in the number of NOS-positive
neurons in CHF compared to control rats (689 ± 76 vs 866 ± 150;
p=0.07, n=5/group), supporting previously published studies. Neurons
containing ENK- or pro-ENK-immunoreactivity were found lateral
to, not within, the PVN; and the numbers of ENK- and pro-ENK-immunoreactive
neurons were similar rats with CHF and sham-operated controls.
These data show that CHF induces changes in the numbers of some
neurochemically-identified populations of PVN neurons but not in
other populations. The populations of PVN neurons that change in
response to CHF are likely to be involved in adaptive mechanisms
that attempt to compensate for the failing heart.
Nitric oxide synthase immunoreactivity
in sympathetic preganglionic neurons
Sympathetic preganglionic
neurons (SPN) are cholinergic and therefore all SPN contain choline
acetyltransferase (ChAT). Several other neurochemical markers
also occur in SPN and one of the most prominent is nitric oxide
synthase (NOS). The proportion of SPN showing NOS-immunoreactivity
has been reported to vary with blood pressure level and published
data suggest that the occurrence of NOS in SPN may also correlate
with the target that they innervate.
To investigate these relationships,
we first evaluated peroxidase immunolabelling for ChAT plus NOS
in spinal cord segments T1-L2 from normotensive Sprague-Dawley
rats (SD) and assessed NOS-immunofluorescence in SPN retrogradely
labelled with cholera toxin B subunit from the adrenal medulla
(AM), or superior cervical (SCG), coeliac (CG) or major pelvic
(MPG) ganglia. We also compared the distributions and numbers of
NOS-positive and NOS-negative/ChAT-positive lateral horn neurons
between SD and spontaneously hypertensive rats (SHR).
In SD, rostrocaudal,
dorsoventral and mediolateral differences occurred in the distributions
of NOS-positive and NOS-negative/ChAT-positive neurons in the intermediolateral
cell column (IML) whereas the two groups were similarly distributed
throughout the central autonomic area (CAA). Amongst the four retrogradely-labelled
populations of SPN, the percentages showing NOS-immunoreactivity
differed (CG-projecting, 54.8 +0.7%; SCG-projecting, 75.3 +1.2%;
MPG-projecting, 89 +1.1% and AM-projecting, 98.6 +0.2%). Within
each retrogradely labelled group of SPN, the NOS-positive proportion
also varied with subnuclear location (e.g., 25.5 +4.0% of CG-projecting
SPN in the CAA vs. 82.7 +7.6% of CG-projecting SPN in the dorsolateral
funiculus). In SHR, the overall distributions of NOS-positive and
NOS-negative/ChAT-positive neurons, their morphologies and their
numbers in T9-11 were the same as in SD.
These results show that
the expression of NOS within SPN varies depending on the target
that they innervate and also on their subnuclear location. Our
data do not support previously published data suggesting that there
are anatomical differences between nitric oxide-synthesizing SPN
in normotensive and hypertensive rats.
Innervation of sacral autonomic neurons
by axons containing corticotropin releasing factor
Brain
neurons immunoreactive for corticotropin releasing factor (CRF)
are involved in regulating micturition. Intrathecal administration
of a CRF antagonist affects voiding and CRF-containing axons occur
in the sacral parasympathetic nucleus, which contains parasympathetic
preganglionic neurons (PPN) controlling bladder function.
To determine
which sacral spinal neurons are innervated by CRF axons , we activated
spinal neurons by evoking micturition reflexes with continuous
infusions of saline in anesthetized male Sprague Dawley rats and
perfused at 2 hours after the commencement of the infusion. Transverse
sections of L5-S2 were stained with a black peroxidase reaction
product to reveal immunoreactivity for CRF and Fos, a marker of
neuronal activation. A brown product was used to localize immunoreactivity
for choline acetyltransferase (ChAT), nitric oxide synthase (NOS)
or cholera toxin B subunit (CTB) retrogradely transported from
the major pelvic ganglion, all of which mark PPN. Neurons with
Fos-immunoreactive nuclei were concentrated in the sacral parasympathetic
nucleus and Lissauer’s tract. Fos-positive
neurons also occurred in lamina X above the central canal. Most
Fos-positive neurons in the sacral parasympathetic nucleus lacked
ChAT, NOS or CTB-immunoreactivity, indicating that they were not
PPN. CRF axons targeted the region of the sacral parasympathetic
nucleus containing PPN and close appositions occurred on Fos-positive
neurons immunoreactive for markers of PPN. CRF axons only occasionally
approached Fos only neurons that lay dorsal to the PPN or occurred
in Lissauer’s tract. CRF axons were rare in lamina X above
the central canal.
These results suggest that CRF axons preferentially
innervate PPN rather than non-preganglionic neurons that are involved
in regulating bladder activity.
Activation of micturition-related sacral
spinal neurons differs between rats with normal and overactive
bladder
Non-voiding contractions are a hallmark of overactive
bladder (OAB), which affects 10-15% of the population in developed
countries. Spontaneously hypertensive rats (SHR) have non-voiding
contractions and reduced bladder capacity; and pharmacological
studies suggest that spinal mechanisms are involved in these changes
in micturition. We investigated whether micturition-related neurons
in the sacral parasympathetic nucleus differed between female rats
with normal and overactive bladders. We induced Fos-immunoreactivity
by evoking micturition reflexes through bladder infusions of saline
under urethane anaesthesia while bladder pressure was being continuously
recorded. Control rats were not infused. Segments L5-S2 from pairs
of infused and control rats were cut transversely and immunoreactivity
for Fos plus choline acetyltransferase (ChAT) were localized immunocytochemically.
Neurons showing immunoreactivity for Fos, ChAT or Fos+ChAT were
counted in each rat. Infused Sprague Dawley (SD) female rats (n=6)
had few non-voiding contractions whereas 3/6 infused SHR females
and 4/6 infused Wistar-Kyoto (WKY) had many non-voiding contractions.
Large numbers of Fos-immunoreactive neurons were present in all
infused SD. In infused SHR and WKY, Fos-positive neurons were very
variable in number but consistently less numerous than in infused
SD. Control SD rats had many fewer Fos-immunoreactive neurons than
infused SD rats. In contrast, in SHR and WKY, the numbers of Fos-positive
neurons in pairs of infused and control rats were often similar.
In infused rats that had more Fos-positive neurons than control
rats, Fos-positive neurons occurred mainly in and dorsal to the
parasympathetic nucleus; double-labelled neurons were concentrated
in the sacral parasympathetic nucleus and around the central canal.
Statistical analysis showed that the number of Fos-positive PPN
was inversely correlated with the number of voiding and non-voiding
contractions that occurred in the first 30 minutes of the infusion
period.
These findings indicate that neural control of micturition
differs between rats with OAB and rats with normal bladder function.
Furthermore, since many WKY appear to have OAB, they are not appropriate
controls for studies on bladder function in SHR.
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