Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels.

Suh BC, Inoue T, Meyer T, Hille B.

Science. 2006 Dec 1;314(5804):1454-7. PMID: 16990515

Objective:   One of the major signaling pathways near the cell membrane involves the activation of phospholipase-C to cleave phosphotidylinositol 4,5-bisphosphate (PIP2) in to diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3).  It should, however, be noted that PIP2 does not serve only as a reserve to be cleaved as needed, but also has the important function of stabilizing the “open” state of KCNQ potassium channels. One challenge researchers have faced is in examining the effects of PIP2 depletion on KCNQ channels without activating the downstream effects that result from activating the entire cascade.  In this paper, a novel method of PIP2 depletion was developed that did not result in production of DAG or IP3.

Methods:    NIH3T3 fibroblast cells were transfected with KCNQ channel subunits and synthetic fusion constructs which would allow for the relocation of a cytosolic PIP2-(5)-phosphatase to the membrane in response to the application of a rapamycin analog, iRap.  The phosphatase was fused to a CFP reporter.  The cells also were made to express a membrane-localized YFP-bound PIP2 reporter that would relocate to the cytosol after cleavage of the 5-phosphate.  Cells were monitored for changes in fluorescence in response to drug exposure and currents were recorded by whole-cell voltage clamp.

Results:

·         When exposed to iRap, cytosolic phosphatase-CFP successfully relocated to the membrane while the YFP-PIP2sensor relocated to the cytosol.  When a phosphatase-dead or phosphatase-missing construct was used, it relocated to the membrane but the PIP2 sensor did not relocate to the cytosol.

·         When using a drug to activate PLC, they could see YFP-PIP2sensor relocation without the CFP-phosphatase relocation.  This relocation was reversible when the drug was washed out.  Under iRap and PLC-activation conditions, potassium currents were inhibited by PIP2 depletion.

·         Under conditions of PLC inhibition, an upstream PLC activator had no effect while iRap did, demonstrating that iRap did not have crossover effects on the PLC pathway.  This was confirmed by use of a DAG indicator and a calcium sensor (indicative of IP3 production).

·         Another construct was used in which iRap activated a kinase to regenerate PIP2 from other phosphatidyl inositols.  This caused PLC activation to have a smaller effect on KCNQ currents.

Conclusions:

·         KCNQ channels require a constant supply of PIP2 to maintain the stability of their “open” conformation.

·         New fusion constructs allow depletion of PIP2 without generation of DAG or IP3

·         New constructs also allow the regeneration of PIP2 faster than a cell could normally do it.

 

-DH

In vivo patch-clamp recording from locus coeruleus neurones in the rat brainstem.

Sugiyama D, Hur SW, Pickering AE, Kase D, Kim SJ, Kawamata M, Imoto K, Furue H.

J Physiol. 2012 May 1;590(Pt 10):2225-31. PMID: 22371480

 

Objective:   The locus coeruleus (LC) contains a large number of noradrenergic neurons that project throughout the brain and modulate activity in a number of centers that control things such as cardiorespiratory activity, arousal, etc.  In order to examine the cellular mechanisms involved in controlling the adrenergic neurons of the LC, this paper used blind in vivo whole-cell patch-clamp to measure and manipulate action potentials caused by hindlimb pinch.

Methods:    Sprague-Dawley rats were anaesthetized and artificially ventilated by tracheostomy.  Extracellular (EC) recording was done with a tungsten microelectrode lowered in to the LC through a burr hole in the skull.  Rats had a portion of the cerebellum removed in order to expose the brainstem.  Patch electrodes were filled with artificial intracellular solution and lowered through the dorsal surface of the pons in to the LC.  Cells were identified for patch clamping through their cell-attached spontaneous firing pattern and response to toe pinch.  Some patched cells were labeled with neurobiotin for later analysis.

Results:

·         EC recording of cells showed a spontaneous firing rate of ~7.4Hz which transiently increased following a contralateral toe pinch.  These characteristics were similar to what was seen using cell-attached and whole- recordings.  This demonstrates a good membrane seal and intracellular solution.

·         Toe pinch under voltage-clamp did not show fast excitatory post-synaptic currents, but a longer slow inward current.

·         Blockade of potassium channels with cesium allowed excitatory and inhibitory post-synaptic currents (EPSCs and IPSCs, respectively) to be isolated.  Drugs could be applied to the surface of the pons, which would affect the neurotransmission of the LC.  EPSCs were blocked with CNQX, an AMPA-receptor-antagonist, but recovered after wash-out.  IPSCs were reversibly blocked by the GABA-A antagonist, bicuculline.

Conclusions:

·         Whole-cell patch clamp can be performed on brainstem neurons in vivo.

·         This preparation allows for pharmacological investigations of the cellular mechanisms of neurotransmission in an in vivo state.

 

 

-DH

Crosslinking the ligand-binding domain dimer interface locks kainate receptors out of the main open state.

Daniels BA, Andrews ED, Aurousseau MR, Accardi MV, Bowie D..

J Physiol. 2013 Aug 15;591(Pt 16):3873-85. PMID: 23713029

 

Objective:   The simplified way of viewing ionotropic glutamate receptors is that they are in their closed conformation until the ligand is bound, at which time they open and allow full ion conductance.  However, recent evidence has demonstrated that a variety of factors, such as the presence of protons or the concentration of ligand at multiple ligand-binding domains (LBDs), may actually lead to a number of permeability states.  In this paper, the ionotropic kainate-type glutamate receptors (KARs)were examined for changes in conductance under conditions of dimer crosslinking through covalent disulfide bonds.

Methods:    tsA201 cells were transfected with mutant/wild type genes encoding the GluK2 subunit.  Outside-out and inside-out recordings were performed on membrane patches to examine GluK2 responses and single-channel events, respectively.

Results:

·         In response to 10mM glutamate, the peak response in a double cysteine mutant in which bonds are known to form was less than 1/10 that of wild type GluK2.  Single mutants had responses either similar to the wild type or completely absent. This may be due to a masking of the LBD or changes in the channel conductance and desensitization.  Analysis of single channel events and computer modeling demonstrates that crosslinking the LBD does not block desensitization by keeping the channel open, but may block ligand binding and block desensitization

·         Analysis of membrane noise, the double mutant was calculated to have a lower conductance than wild type channels.  This was due to a lack of high-conductance open states seen in wild type channels, presumably due to the crosslinking.

·         By summing 3 and 4 exponential components, it was shown that the wild-type channels have shorter shut-states than the mutants do.

Conclusions:

·         Crosslinking LBDs of KARs must alter multiple aspects of channel function

·         Breaking the disulfide bonds doesn’t restore wild-type function, so other effects must also occur

·         Different glutamate receptors may respond differently to LBD crosslinking

 

-DH

AT₁ angiotensin II receptor and novel non-AT₁, non-AT₂ angiotensin II/III binding site in brainstem cardiovascular regulatory centers of the spontaneously hypertensive rat.

The authors investigated two aims in this study.  The first aim is to compare AT1 receptor binding in the RVLM and other cardiovascular control regions between SHR and WKY.  They hypothesize that AT1 receptors are more abundant in the RVLM of SHR. The second aim is to compare the relative expression of the binding sites between SHR and WKY in the DMM, RVLM and CVLM. They hypothesize that the binding site is differentially expressed in hypertensive rats.  Similar to their previous studies the authors used autoradiography to determine the receptor binding. They found an increased AT1 receptor binding in DMM (dorsalmedial medulla), CVLM and RVLM of SHR compared to WKY.  However, non-AT1, non-AT2 receptor, Ang II and Ang III binding sites were decreased in the RVLM and DMM of SHR. The increased AT1 receptor binding the RVLM could contribute to the hypertension of SHR.  The authors believe that reduced expression of non-AT1 and non-AT2 binding sites in the RVLM may be involved in the reducing the blood pressure through its interaction with Ang II and Ang III.

-Madhan

Water deprivation increases angiotensin-converting enzyme but not AT1 receptor expression in brainstem and paraventricular nucleus of the hypothalamus of the rat

The authors investigated whether water deprivation in normotensive rats produce an upregulation of the central renin-angiotensin system in the areas of the brain that are involved in blood pressure control especially PVH and RVLM. They measured AT1 receptor expression in a number of regions in the brain using quantitative AT1 receptor autoradiography.  Similarly angiotensin converting enzyme (ACE) was also measured in the some of these regions using a similar technique. They found that ACE expression was increased in brain regions that are important for blood pressure control. However there was no difference in AT1 receptor expression in the brain regions tested between dehydrated rats and water-replete controls except in subfornical organ (SFO), a circumventricular lacking blood brain barrier. This study partially proves that during dehydration the central RAS could maintain blood pressure through increasing the expression of ACE. Similarly increased AT1 receptors in the SFO indicate an increased response to Ang II, because of the lack of blood brain barrier in the SFO. SFO sends excitatory projections to the PVN. The neurons form the PVN projects to RVLM, which then projects to the intermediolateral cell column of the spinal cord to activate the preganglionic neurons of the sympathetic nervous system.  The authors believe during water deprivation, the above mentioned pathway could be involved in the maintenance of normal blood pressure.  

-Madhan

Role of angiotensin-(1-7) in rostral ventrolateral medulla in blood pressure regulation via sympathetic nerve activity in Wistar-Kyoto and spontaneous hypertensive rats.

Nakagaki T, Hirooka Y, Ito K, Kishi T, Hoka S, Sunagawa K.

Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.

Clin Exp Hypertens. 2011;33(4):223-30. doi: 10.3109/10641963.2011.583967.

   

The Renin-Angiotensin System (RAS) modulates blood pressure, this study focuses upon Angiotensin-(1-7) in the RVLM. The objective of the present study was to determine whether Ang-(1-7) in the RVLM contributes to the maintenance of BP in the rat model of hypertension. Male Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) used. SHRs and WKYs monitored for BP, HR, and renal sympathetic nerve activity (RSNA). Rats injected with Ang (1-7), Mas receptor antagonist A-779, and ACE2 inhibitor DX600. They also performed Western Blot analysis for Mas receptors in SHR and WKY. Unilaterial Ang-(1-7) microinjection inducted a significantly greater increase in MAP in SHR than WKY. Bilateral A-779 microinjection induced a significantly greater decrease in AP and renal sympathetic nerve activity in SHR than WKY. Bilateral DX600 microinjection induced a significantly greater decrease in AP in SHR than in WKY. These results suggest that endogenous Ang-(1-7) in the RVLM contributes to maintain AP and renal sympathetic nerve activity in both SHR and WKY, and that its activity is likely enhanced in SHR. We could probably do a similar study in WR and SEDs, however, it would probably just lead to a somewhat similar result to this SHR v. WKY study. This study suggests that there is a greater deal of sympathoexcitation for any given input in the case of non-normotensive conditions.

Posted by Max 

Anteroposterior distribution of AT(1) angiotensin receptors in caudal brainstem cardiovascular regulatory centers of the rat.

The purpose of this study is to investigate the density of AT1 receptors in the RVLM, CVLM and DMM (Dorsomedial medulla) regions of the rats. The authors used adult female retired breeder Wistar rats to evaluate AT1 receptor binding along a 2.7mm length of medulla.   They used receptor binding autoradiography to visualize AT1 receptor binding in the above mentioned regions. The significant finding from the present study is that they showed AT1 receptor binding in a rostro-caudal plane extending from CVLM to RVLM. The density of AT1 receptor is fairly constant along the rostro-caudal extent.  They found that AT1 receptor binding density (Bmax) was lower in CVLM and RVLM than DMM.  The dissociation constant (KD) of AT1 binding sites in the RVLM and CVLM is lower indicating higher affinity i.e. neurons in the RVLM and CVLM are more sensitive to Ang II compared to DMM.

-Madhan 

Water Deprivation Activates a Glutamatergic Projection from the Hypothalamic Paraventricular Nucleus to the Rostral Ventrolateral Medulla

SEAN D. STOCKER,1,2 JOHNNY R. SIMMONS,3 RUTH L. STORNETTA,3*

GLENN M. TONEY,2 AND PATRICE G. GUYENET3

1Department of Physiology, University of Kentucky College of Medicine,

Lexington, Kentucky 40536-0298

2Department of Physiology, University of Texas Health Science Center,

San Antonio, Texas 78229

3Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908

THE JOURNAL OF COMPARATIVE NEUROLOGY 494:673–685 (2006)

This study examined compensatory mechanisms within the medullary circuit in response to water deprivation. As we already know (or should know) the PVN generally plays a greater role in cardiovascular regulation under non-normotensive conditions. This study sought to see the role of PVN in the case of water deprivation, particularly in regards to its projections to RVLM. They sought to determine whether the PVN-RVLM projections activated by water deprivation were glutamatergic and/or contained vasopressin or oxytocin. Adult male Sprague-Dawley rats were injected with several retrograde tracers into both their PVN and RVLM, as a means to track the projections between the two areas. They were first microinjected with CTB into their RVLM (wow that’s cool), and then exposed to a water deprivation protocol or not (control). Following a period of about two weeks these rats were sacrificed and then perfused. After the brain tissue was retrieved and fixed, they conducted in-situ hybridization to quantify mRNA in PVN to determine whether or not PVN had glutamatergic or GABAergic projections to RVLM. As a result of measuring the VGLUT2 in PVN, they were able to deduce that PVN actually had glutamatergic projections to RVLM, and were particularly activated during water deprivation. This once again suggests that PVN plays a role in non-normotensive conditions and that it may project glutamate onto RVLM in the case of hypertension and other CVD.

Posted by Max 

Non-NMDA receptors in the rostral ventrolateral medulla mediate somatosympathetic pressor responses.

James M. Kiely and Frank J. Gordon

Journal of the Autonomic Nervous System, 43(1993) 231-240

      This paper studied the role of excitatory amino acid receptors in the rostral ventrolateral medulla (RVLM) in mediating somatosympathetic pressor response (SPR). This SPR is prompted by sciatic nerve stimulation, something that I do within my experiments.  Female Sprague-Dawley rats were anesthetized with urethane and then followed by microinjection protocol of NMDA (D-AP7) or non-NMDA (DNQX) antagonists. By differentially blocking both NMDA and non-NMDA receptors throughout several protocols, they found that the non-NMDA receptors were the receptors actually mediating the SPR. This paper offers one of the earliest pieces of evidence of interneurons in RVLM, due to the fact that D-AP7 prevented the blockade and reversal of SPR by DNQX. The observations of this paper suggest the possibility of sympathoinhibitory interneurons within the RVLM which can be stimulated by activation of NDMA receptors. A few issues I have with this paper is the fact that they were able to illicit such high pressor responses from sciatic nerve stimulation (about 45 mmHg) while I can only increase pressure by about (15 mmHg) with a similar stimulation.

Posted by Max 

Brain Sources of Inhibitory Input to the Rat Rostral Ventrolateral Medulla

Belinda R. Bowman, Natasha N. Kumar, Sarah F. Hassan, Simon McMullan, and Ann K. Goodchild*

Australian School of Advanced Medicine, Macquarie University, 2109, NSW Australia

The RVLM receives inhibitory and excitatory input in order to maintain homeostasis.  However the focus of this article was to investigate sources of GABAergic and preproenkephalin input to the RVLM. They injected CTB into the RVLM.  They used in situ hybridization in order to identify GABAergic and preproenkephalin. They found that there are multiple inhibitory inputs going to RVLM are either GABAergic and/ or encephalin but the most interesting finding 17% of gabaergic inputs come from CVLM.  However CVLM is the major source of tonic GABAergic input to RVLM.  

Angiotensin-(1-7) in the Rostral Ventrolateral Medulla Modulates Enhanced CardiacSympathetic Afferent Reflex and Sympathetic Activation in Renovascular Hypertensive Rats

Peng Li, Hai-Jian Sun, Bai-Ping Cui, Ye-Bo Zhou and Ying Han
Hypertension. 2013;61:820-827; originally published online February 19, 2013;
doi: 10.1161/HYPERTENSIONAHA.111.00191 The purpose of this was to determine whether ang 1-7 in the RVLM is involved in the enhanced CSAR in renovascular hypertension.  Secondly to determine whether cAMP – PKA pathway is playing a role in mediating the ang1-7 effects on the CSAR in the RVLM of renovascular hypertensive rats.   What they determined from the study is that in the renovascular hypertensive model not only demonstrate enhanced RSNA and map responses; they also found enhanced mas receptor expression and also that by blocking Ang 1-7 with the antagonist A-779 that the CSAR is blocked. Finally to determine if CAMP-PKA pathway is involved they gave inhibitors to PKA and showed that greater responses in RSNA and MAP were greater in the hypertensive model. The same result was demonstrated with the cAMP antagonist except that with pretreatment of cAMP the CSAR response was not abolished however it was with the PKA antagonists.  cAMP levels were also enhanced in response to activation of the CSAR.   They basically showed that Ang 1-7 in the RVLM enhances the CSR along with RSNA and MAP. This is done by acting on MAS receptors which ultimately lead to activation of the cAMP-PKA pathway.

Exercise Training Lowers the Enhanced Tonically Active Glutamatergic Input to the Rostral Ventrolateral Medulla in Hypertensive Rats

The authors tested how low intensity exercise affects the glutamatergic input to the RVLM in spontaneously hypertenive rats. They used WKY and SHR and trained them in treadmill for a period of 12 weeks. Citrate synthase concentration was measured as a measure of exercise training efficacy.  Microinjections were performed in the RVLM to study the changes in BP, HR and RSNA. Blood pressure for the time course experiment were performed using tail-cuff method. Western blotting was performed in RVLM punches to detect the changes in protein levels of vesicular glutamate transporter 2 (vGLUT2). High-performance liquid chromatography (HPLC) was used to measure the glutamate concentration. Data was analysed by a two-way ANOVA followed by post hoc with Student Newman-Keuls. Exercise training decreased the baseline BP and RSNA in SHRs. Execrise training attenuated the decrease in BP, HR and RSNA evoked by blockade of glutamate receptors in the RVLM of SHRs. Exercise training in the SHRs decreased the concentration of glutamate and protein expression of vGLUT2. The overall conclusion is that exercise training lowers the tonically active gluamatergic input in the RVLM of SHRs.

-Madhan

Role of the Rostral VentrolateralMedulla in the Arterial Hypertension in Chronic Renal Failure

Adriana P. Castilho Dugaich,1 Elizabeth B. Oliveira-Sales,1 Nayda P. Abreu,
Mirian A. Boim,2 C´assia T. Bergamaschi,1 and Ruy R. Campos3 
 A component of chronic renal failure is high circulating angiotensin II and this leads to an increase in NAD(P)H oxidase , which is responsible for the production of superoxide. They wanted to quantify the subunits of NAD(P)H which are NADPH p47phox and gp91phox in CRF . They also looked at AT1 expression since Ang II is involved. In order to further investigate the role of oxidative stress and AT1 in hypertension in CRF, they injected tempol into the RVLM and the AT1 antagonist-candesartan. They also investigated glutamate and GABA sensitivity in CRF. mRNA expression for NADPHp47phox and gp91phox in the brainstem were greater in the CRF when compared to the control, however the expression of AT1 was down regulated in the CRF group. Tempol responses in rvlm were greater in the CRF and the control group actually had no response to tempol, further demonstrating that CRF leads to superoxide production in the RVLM. Responses to AT1 Ang II antagonist candesartan were not significantly different between groups. Glutamate responses were attenuated in the CRF group when compared to the control. GABA responses were greater in the CRF group compared to the control group. The data suggest that oxidative stress is contributing to the development of hypertension in CRF animals.

Our own Dr. Mueller has created this slide summarizing the major results of the 2005 and 2006 papers by Nelson et al.  Also included in this image are results from the 2010 paper, soon to be make an appearance on the Muller lab blog.

Dibs
-DH

Reversibility of exercise-induced dendritic attenuation in brain cardiorespiratory and locomotor areas following exercise detraining.

Nelson AJ, Iwamoto GA.

J Appl Physiol. 2006 Oct;101(4):1243-51.PMID: 16794024

Objective:   The cardioprotective effects of voluntary exercise in animal models are known to decline in as little as two weeks after cessation of activity.  Having previously shown that training, or being physically active, results in morphological changes in the dendrites and somata of neurons found in cardiorespiratory-locomotor centers, the authors of this paper used Golgi-Cox staining to examine changes in dendritic morphology in areas related to cardiorespiratory-locomotor activity after “detraining,” or cessation of physical activity.

Results:

·         The 56-day trained rats ran significantly more per week than the 120-day trained rats, possibly due to differences in humidity between test groups.
·         Untrained and detrained animals at 120 days did not have significant differences in mass, body fat percentage, oxygen consumption, heart rate, heart weight, or heart weight to body ratio. 56-day trained animals had significantly higher oxygen consumption as well as significantly lower weight and heart weight to body weight ratio when compared to untrained animals.
·         Untrained and detrained rats at 120 days had no observable differences in dendritic fields or neuron size in the periaqueductal gray, posterior hypothalamic area, nucleus tractus solitarioius, or cuneiform nucleus.  Untrained animals had significantly more dendritic branching in the cuneiform nucleus and nucleus tractus solitarious than trained animals when measured at day 56.
·         The cuneiform nucleus of trained rats showed significantly smaller somata than trained rats at day 56 (consistent with previous findings) but the nucleus tractus solitarious did not.

Conclusions:

·         The morphological changes in neurons of the cardiorespiratory-locomotor centers effected by 56 days of voluntary activity are not present in rats that experience 70 days of activity followed by 50 days of inactivity.
·         The systemic cardioprotective effects of activity that decline following periods of inactivity are reflected in changes to neuronal morphology in cardiorespiratory-locomotor centers of the brain.

Methods:   Rats were divided in to three groups.  “Trained” animals were allowed free access to run on a wheel until they were killed after 56 days.  “Detrained” animals were allowed to run from days 1 to 70, but not after, and then killed at day 120. “Untrained” rats were denied access to a wheel entirely and killed at either day 56 or 120.  Animals were given two familiarization trials on a treadmill with increasing speed 4-6 days before a testing trial, rats were placed on a treadmill and monitored for maximum oxygen uptake and carbon dioxide production.  One week after the oxygen consumption test, rats were monitored for resting heart rate, killed, and had their hearts and brains removed for examination.  Brains were immersed in Golgi-Cox impregnating solution for 2 weeks, frozen, cut in to 200um sections, and stained for examination of cell morphology.  Neurons in regions of interest were traced/drawn using camera lucida and examined for the number of dendritic branch points and their distances from the soma.

 -DH

Neuroplastic adaptations to exercise: neuronal remodeling in cardiorespiratory and locomotor areas.

Nelson AJ, Juraska JM, Musch TI, Iwamoto GA.

J Appl Physiol. 2005 Dec;99(6):2312-22. PMID: 16123206

Objective:   Voluntary exercise in animal models has been shown to cause changes in neuronal morphology and function as well as changes in the function of peripheral organs such as decreases in heart rate and blood pressure.  In this paper, Golgi-Cox staining was used to examine changes in dendritic morphology in six areas related to cardiorespiratory-locomotor activity and in one area of the cerebral cortex in active and inactive animals.

Results:

·         In active animals, running increased from week 1 to week 8 before declining until week 14, where it remained level until week 18.  No differences were found between rats of the 85 (week 12) and 120 (week 18)day groups when examined for heart rate, dendritic branching, maximum oxygen consumption, fat-free mass.  Data from the two groups were pooled.
·         Active animals had significantly lower body weight, maximum oxygen uptake, and heart rate and significantly higher heart weight, heart weight to body mass ratio, and heart weight to fat-free mass ratio when compared to inactive animals.
·         Significantly more dendritic branching was seen within the first 120um from the soma in the periaqueductal gray, posterior hypothalamus, cuneiform nucleus, and nucleus tractus solitarious, but not in the rostral ventrolateral medlla (RVLM),  nucleus cuneatus, or the hindlimb area of the cortex when comparing these areas in active animals to inactive animals. The somata of neurons in the periaqueductal gray and cuneiform nucleus were significantly smaller in active animals than those in inactive animals.

Conclusions:

·          Exercise results in morphological changes in neurons of some cardiorespiratory-locomotor centers as demonstrated by reductions in dendritic branching and soma diameter.
·          The RVLM did not show changes, possibly because of its involvement in other functions such as sympathoexcitation and because its continuous activity in blood pressure maintenance results in a stable morphology.

Methods:   Active rats were allowed access to a running wheel and monitored for activity while inactive rats were not.  After 85 or 120 days, all animals were given two familiarization trials on a treadmill with increasing speed.  During testing trials, rats were monitored for oxygen uptake and carbon dioxide production.  Two days after, rats were monitored for resting heart rate, killed, and had their hearts and brains removed for examination.  Brains were immersed in Golgi-Cox impregnating solution for 2 weeks, frozen, cut in to 200um sections, and stained for examination of cell morphology.  Neurons in regions of interest were traced/drawn using camera lucida and examined for the number of dendritic branch points and their distances from the soma.

 -DH

In vivo auditory brain mapping in mice with Mn-enhanced MRI.

Nat Neurosci. 2005 Jul;8(7):961-8. PMID: 15924136

Yu X, Wadghiri YZ, Sanes DH, Turnbull DH.

The objective in this study was to develop a noninvasive imaging method of imaging changes in activity of murine brainstem nuclei related to brain development and hearing loss.  Using manganese enhanced MRI (MEMRI) shows differences in Mn²⁺ uptake caused by changes in neuronal activity.  This allows the measurement of changes in activity that result from hearing loss at different stages in neurodevelopment.

Results:

·         Mn²⁺ was enhanced in ventricles within 2hrs, but cleared over 24hrs concomitant with increased Mn²⁺ in parenchyma leading up to the 24hr point.  The greatest increase in signal was seen in the olfactory bulbs, hippocampus, midbrain, and cerebellum.
·         Animals with bilateral CHL showed significantly lower Mn enhancement of the auditory brainstem nuclei than control animals.  There were no significant differences in non-auditory areas.
·         Animals with unilateral CHL showed significant Mn²⁺ enhancement in the cochlear nucleus (CN) corresponding to the functional ear compared to the CN of the non-functional ear, which were similar to the differences in enhancement between experimental and control animals.  The enhancement also appeared in the inferior colliculi (IC) contralateral to the CN with Mn²⁺ enhancement, in agreement with the known axonal projections.
·         MEMRI was able to demonstrate frequency-dependent tonotopic enhancement in regions of the IC  that was in agreement with existing data from electrophysiological studies.
·         At the 6 week point, CHL induced at p10 (before the onset of hearing) resulted in decreased enhancement of both the CN and the IC, whereas CHL induced at p21 showed changes in the CN but not the IC.  This indicates that early hearing loss leads to persistent changes in auditory brain function.

Conclusions:

·         MEMRI has spatial resolution sufficiently high enough to allow for tonotopic mapping of the IC
·         MEMRI can demonstrate changes in neurodevelopment and function.
·         This method shows MEMRI can be done at non-toxic doses and less invasively than previous methods (no cannula or disruption of blood brain barrier) in order to measure activity-dependent Mn²⁺ uptake.

Methods: Unilateral or bilateral conductive hearing loss (CHL) was induced in mice at postnatal day 10, 21, or week 6 via puncture of the tympanic membrane and removal of the malleus. MnCl₂ was administered via IP injection at 0.4mm/kg as this dose was shown to be below the threshold of neurotoxicity. Mice were exposed to 24 hours of sound at frequencies within the range of their hearing. MRI data was then acquired and analyzed to find regions of interest as defined by and increased signal intensity due to Mn²⁺uptake. Signal intensity was normalized to the signal of the caudate putamen, which was a region unaffected by sound stimulation.

-DH

In vivo 3D MRI staining of mouse brain after subcutaneous application of MnCl2.
Magn Reson Med. 2002 Nov;48(5):852-9. PMID: 12418000
Watanabe T, Natt O, Boretius S, Frahm J, Michaelis T.

The objective of this paper was to examine the feasibility of using Mn²⁺ as an MRI contrasting agent without the need to disrupt the Blood Brain Barrier as has been done in other studies.  This would allow visualization of brain structures after systemic application of Mn²⁺, without the need for catheterization of the carotid artery to deliver mannitol solution for disruption of the BBB.

Results:

·         Areas with direct access to systemic circulation (anterior pituitary, choroid plexus) show strong increase (200%) at 6hr point.  Areas behind blood brain barrier (olfactory bulb, hippocampal CA3 region) take longer (24hrs) and show a weaker increase (10-25%)
·         Observed tract-like structures in the olfactory bulb – interpreted as evidence for axoplasmic transport of Mn²⁺
·         Saw bright superficial layers in the cerebellum similar to purkinje cell layers and dark deep layers representative of white matter.
·         Saw an increase in CA3 (input region), but not in CA1 (output region)

Conclusions:

·         MRI enhancement of selective areas is by differential uptake of Mn²⁺ through Ca²⁺ channels during action potentials.  Differences in Mn²⁺ content reflect differences in neuron activity during the time after the MnCl₂ injection
·         Differences may also rely on differential expression of enzymes that utilize Mn²⁺ (e.g. glutamine synthetase contains 8 Mn²⁺ ions and contains ~80% of Mn in the brain, SNR was higher in CA3 which is known to have high expression of manganese superoxide dismutase and lower in CA1 that doesn’t)

Methods:  Mice were given subcutaneous injections of MnCl₂.  After IP anesthesia, MRI was performed at 0, 6, 24, and 48hrs in order to compare the Mn²⁺ enhancement in 13 different regions of the brain.  Evaluation of Mn²⁺ enhancement was done by comparing the Signal to Noise ratio, where SNR=(Signal/StdDev)

-DH

PACAP causes PAC1/VPAC2 receptor mediated hypertension and sympathoexcitation in normal and hypertensive rats.

Am J Physiol Heart Circ Physiol. 2012 Oct 1;303(7):H910-7. 
Farnham MM, Lung MS, Tallapragada VJ, Pilowsky PM.

"Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuropeptide that plays and important role in hypertension and stress responses". 

The authors proposed an idea that PACAP acts as a sympathoexcitatory agent in the RVLM of normotensive and hypertensive rats. The had 3 specific aims for the experiment and tested them using different mechanisms as in described in the above illustration. The major findings of this study is that injection of PACAP in the RVLM caused an increase in splanchnic SNA, heart rate and MAP in SHR, WKY and SD rats. Pretreatment with PAC1/VPAC2 receptor antagonist PACAP (6-38) attenuated this response but did not produce any changes when injected alone suggesting that PACAP receptors are not tonically active. PACAP (6-38)  in the RVLM produced no changes in sSNA or MAP in the SHR suggesting they may not play a role in maintaining hypertension in SHR.

-Madhan

Exercise training causes sympathoinhibition through antioxidant effect in the rostral ventrolateral medulla of hypertensive rats

Clin Exp Hypertens. 2012;34(4):278-83.
Kishi T, Hirooka Y, Katsuki M, Ogawa K, Shinohara K, Isegawa K, Sunagawa K.

The authors were interested to know whether exercise training affects sympathetic nerve activity through central mechanisms in stroke-prone spontaneously hypertensive rats (SPSHR). They performed a series of experiments to determine the role of oxidative stress and angiotensin II in this pathway. First, they implanted radio-telemetry system to measure mean arterial pressure and heart rate in both SHRSP and Wistar-Kyoto (WKY) rats. The animals were seperated into 4 groups exercise trained (Treadmill) and non exercise trained for 28 days. After 28 days, urinary norepinephrine was measured as an indicator of sympathetic nerve activity. They measured conscious baroreflex sensitivity by spontaneous sequence method. Thiobarbituric acid-reactive substances were measured from RVLM punches as an indicator of oxidative stress. Tempol, a superoxide dismutase mimetic and angiotensin II were microinjected into the RVLM to determine the pathway. The overall findings are that exercise training in SHRSP caused sympathoinhibition and improved baroreflex sensitivity and reduced oxidative stress through blocked AT1R in the RVLM.

-Madhan