This article gave an overview of the 3 main nuclei that play a role in the baroreceptor reflex. It discussed the different neurotransmitters that participates in the modulation and transmission of the signals from one nuclei to another. When I think about the NTS, CVLM and the RVLM I only think about glutamate and GABA because these are the primary players. I never think about the other transmitters that bind to their specific receptors and effect the presynaptic release of glu and GABA . Some of the information that was presented about the RVLM was new to me. For instance I did not realize that there was a respiratory region within the RVLM. The respiratory region can also affect SNA.
The most interesting statement mentioned in the paper was whether the RVLM was a network or pacemaker. I know that it has netwrok characteristics because inputs from other region can modulate the activity of the RVLM. As for the Pacemaker, I can't recall any papers that have said this. Maybe I have read papers and I never really thought about the RVLM in these terms. Is the RVLM a network or Pacemaker? Can someone give me some input or may even a link to an article that may help clarify this?
Thursday, June 30, 2011
Friday, June 24, 2011
Nucleus tractus solitarius and control of blood pressure in chronic sinoaortic denervated rats
It has been shown by previous laboratories, that chronic sinoaortic denervated rats blood pressure return to normal ranges. However, initially, they show elevated blood pressure. This article investigated whether the NTS is playing a role in the tonic regulation of blood pressure in chronic SAD rats because the blood pressure returns to a normal range after a couple weeks. They believed that it could be possible that there are cardiopulmonary afferents traveling to the NTS, in the absence of the barorecptor afferents to the NTS, the cardiopulmonary afferents are then responsible for relaying information about blood pressure to the NTS so that tonic control of blood pressure is still possible.
The investigators did a couple of interesting things, they microinjected muscimol or lidocaine into the NTS bilateral, the rats were anesthetized for this experiment. For another experiment,by using electrical current they were able to lesion the NTS in chronic SAD and control animals. After the NTS lesion was completed the rats were allowed to recovery from the surgery and the recording of the new AP and HR was done while the animal was conscience animal ( only waited 1 hour after the NTS lesion surgery). In both the control and chronic SAD you would expect an increase in AP and in HR as a response for both procedures if their hypothesis is correct. This was not what they saw in the Chronic SAD , in both of the experiments the chronic SAD had no response, so AP and HR did not change. However, they did see the increase in the AP and in HR in the control rat, along with an increase in the vasopressin concentration in the plasma ( only tested VP before and after applying an electric current to lesion NTS). In order to make sure that the Chronic SAD rats were actually able to secrete VP, they tested all the animals to see their response to hypertonic saline. They should release VP in response to hypertonic saline and that is the result they saw. So there was nothing wrong with the chronic SAD rats ability to secrete VP.
So their findings showed that the NTS is not necessary for tonic control of AP and HR in chronic SAD. There must be another area responsible for this regulation when the barorecptors inputs are abolished or it could be possible that the vessels might have so mechanism that allows for tonic control of AP. They also showed that the chronic SAD rats still had some cardiopulmonary bararecptor reflexes, however ,this does not contribute to the tonic control of AP that is seen in the chronic SAD rats.
The investigators did a couple of interesting things, they microinjected muscimol or lidocaine into the NTS bilateral, the rats were anesthetized for this experiment. For another experiment,by using electrical current they were able to lesion the NTS in chronic SAD and control animals. After the NTS lesion was completed the rats were allowed to recovery from the surgery and the recording of the new AP and HR was done while the animal was conscience animal ( only waited 1 hour after the NTS lesion surgery). In both the control and chronic SAD you would expect an increase in AP and in HR as a response for both procedures if their hypothesis is correct. This was not what they saw in the Chronic SAD , in both of the experiments the chronic SAD had no response, so AP and HR did not change. However, they did see the increase in the AP and in HR in the control rat, along with an increase in the vasopressin concentration in the plasma ( only tested VP before and after applying an electric current to lesion NTS). In order to make sure that the Chronic SAD rats were actually able to secrete VP, they tested all the animals to see their response to hypertonic saline. They should release VP in response to hypertonic saline and that is the result they saw. So there was nothing wrong with the chronic SAD rats ability to secrete VP.
So their findings showed that the NTS is not necessary for tonic control of AP and HR in chronic SAD. There must be another area responsible for this regulation when the barorecptors inputs are abolished or it could be possible that the vessels might have so mechanism that allows for tonic control of AP. They also showed that the chronic SAD rats still had some cardiopulmonary bararecptor reflexes, however ,this does not contribute to the tonic control of AP that is seen in the chronic SAD rats.
Tuesday, June 21, 2011
Addressing an ongoing controversy: Do C1 neurons contribute to resting blood pressure?
Control of sympathetic vasomotor tone by catecholaminergic C1 neurones of the rostral ventrolateral medulla oblongata.
This study was designed to investigate the role of rostral ventral medullary catecholaminergic neurons in the control of resting blood pressure. We all know that most of supraspinal resting vasomotor tone comes from some neurons that "live" in the rostral portion of the ventrolateral medulla (RVLM). We also know that many neurons in RVLM are "C1" neurons, that is, they can synthesize catecholamines. Finally, we know that both C1 and non-C1 neurons send axons to sympathetic preganglionic neurons in the spinal cord, with most being C1. At the time of these discoveries it was assumed that C1 neurons were a major source of resting and relfex RVLM-mediated sympathetic activity. However, studies done in the past ten years have been able to investigate the function of C1 neurons by specifically targeting them with antibodies and viruses. Schreihofer and Guyenet showed that when about 75% of C1 neurons were ablated, sympathoexcitatory responses to direct or reflex-mediated stimulation were markedly reduced, but resting tone of SNA and MAP were unaffected. Further work from this lab showed that specific direct activation of C1 neurons produced increases in SNA and MAP. Also, work from another lab showed that specific "re-expression" of angiotensin II receptors in C1 neurons of an angiotensin receptor knockout mouse model restored SNA responses to angiotensin II in the RVLM. Together, these data suggest a prominent role for C1 neurons in SNS responses but doesn't fully address the role of C1 neurons in the maintanance of resting tone.
The very innovative and unique aspect of this study was the system the authors designed to test direct acute inhibition of C1 neurons in vivo. This was accomplished using lentiviruses specific for C1 neurons, but instead of the virus encoding for something that kills the cell, it encodes an inhibitory G protein-coupled receptor that has a specific ligand (allatostatin) which is not present in mammals. After RVLM injection of the virus and a 5 to 6 week recovery period, rats were anesthetized and instrumented to record MAP and renal SNA. They showed convincingly that acute specific inhibition of C1 neurons in vivo causes a reversible fall in MAP and RSNA in animals that recieved the virus but not in sham rats. They then tested the role of C1 neurons in the SNS response to hypercapnia, or high levels of arterial CO2. Interestingly, if C1 neurons were inhibited before experimentally varying CO2 levels, the response was the same as control. If C1 neurons were inhibited during high CO2, the peak response was attenuated. Also, they suggest that blockade of ionotropic glutamate receptors did not affect this phenomenon. I engourage anyone reading this post to look at the results of this second-to-last experiment and decide for yourself what the results suggest. Finally, they used an isolated heart-brainstem preparation to show that C1 neurons generate much of the respiratory-related SNA bursts.
These data contrast with previous work described. However, the authors note an important distinction in the discussion. By using a toxin that killed C1 neurons over the course of days, other vasomotor centers had the opportunity to increase their activity in compensation. Importantly, they showed in this study that the inhibition was immediate and reversible. Additionally, it appears (to me) that glutamatergic inputs on C1 neurons do have some role in the sympathetic response to hypercapnia, although I admit that the results from this study don't clearly suggest whether they do or do not. Finally, C1 neurons clearly are responsible for respiratory-related bursts of SNA in the authors' in situ heart-brainstem prep. They could confirm these results in vivo by performing experiments similar to those from Ann Schreihofer's lab of the past 5 years or so.
Wednesday, June 15, 2011
An overview of laser microdissection technologies
Graeme I. Murray
Acta histochemica 109 (2007) 171-176
In this review, Murray compared and contrasted two methods of laser microdissection, giving pros and cons for laser capture microdissection and laser cutting microdissection. Both have the advantage over traditional microdissection techniques (requiring a scalpel or a fine stainless steel needle), because the older methods of microdissection are "slow, cumbersome, and require considerable dexterity." In addition, using a scalpel or needle makes the tissue sample both hard to acquire and easy to contaminate.
Laser capture microdissection (as we do in our lab) requires a membrane attached to a cap, which is placed over the tissue section and melted with a laser. The melted plastic attaches to the sample, cools within milliseconds, and allows the user to pick up only the small spot of the sample that they have chosen. While this is convenient for collecting samples for use with assays that allow for amplification, like DNA and RNA, it is not very useful for protein assays, as the sample will be quite small. The laser and the temperature changes do not seem to harm the sample, and the settings can be adjusted for use with tissues fixed in various ways.
Laser cutting microdissection was developed more recently and involves using a laser to "draw around" the desired cells, allowing them to detach from the slide. The sample is then dropped or catapulted into a tube for collection. This method does not require costly caps, heating and cooling of a membrane, or contact of any kind with the sample (thus cutting down on opportunities for contamination). It allows for greater precision, as well, because laser capture microdissection has a larger laser diameter with a fixed shape (a circle) for sample collection, whereas laser cutting microdissection allows the user to outline their cells exactly (minimizing the collection of non-target cells that are adjacent to the target cell). However, it works best when only a limited number of cells is needed, as laser capture microdissection allows for a more rapid collection of a large number of cells.
As to the matter of tissue preparation, Murray recommends that the tissue be exposed for as little time as possible to stains or rapid IHC techniques. The biggest takeaway for tissue preparation is to avoid doing anything make the sample less ideal for the assays that one intends to do. For us, because we wish to isolate the RNA, that means using fresh frozen tissue and avoiding letting the sections stay at room temperature for very long, so that the RNA has little opportunity to degrade. Different types of molecular analysis will require different treatment of the tissue, though.
Acta histochemica 109 (2007) 171-176
In this review, Murray compared and contrasted two methods of laser microdissection, giving pros and cons for laser capture microdissection and laser cutting microdissection. Both have the advantage over traditional microdissection techniques (requiring a scalpel or a fine stainless steel needle), because the older methods of microdissection are "slow, cumbersome, and require considerable dexterity." In addition, using a scalpel or needle makes the tissue sample both hard to acquire and easy to contaminate.
Laser capture microdissection (as we do in our lab) requires a membrane attached to a cap, which is placed over the tissue section and melted with a laser. The melted plastic attaches to the sample, cools within milliseconds, and allows the user to pick up only the small spot of the sample that they have chosen. While this is convenient for collecting samples for use with assays that allow for amplification, like DNA and RNA, it is not very useful for protein assays, as the sample will be quite small. The laser and the temperature changes do not seem to harm the sample, and the settings can be adjusted for use with tissues fixed in various ways.
Laser cutting microdissection was developed more recently and involves using a laser to "draw around" the desired cells, allowing them to detach from the slide. The sample is then dropped or catapulted into a tube for collection. This method does not require costly caps, heating and cooling of a membrane, or contact of any kind with the sample (thus cutting down on opportunities for contamination). It allows for greater precision, as well, because laser capture microdissection has a larger laser diameter with a fixed shape (a circle) for sample collection, whereas laser cutting microdissection allows the user to outline their cells exactly (minimizing the collection of non-target cells that are adjacent to the target cell). However, it works best when only a limited number of cells is needed, as laser capture microdissection allows for a more rapid collection of a large number of cells.
As to the matter of tissue preparation, Murray recommends that the tissue be exposed for as little time as possible to stains or rapid IHC techniques. The biggest takeaway for tissue preparation is to avoid doing anything make the sample less ideal for the assays that one intends to do. For us, because we wish to isolate the RNA, that means using fresh frozen tissue and avoiding letting the sections stay at room temperature for very long, so that the RNA has little opportunity to degrade. Different types of molecular analysis will require different treatment of the tissue, though.
Monday, June 13, 2011
Contribution to sympathetic vasomotor tone of tonic glutamatergic inputs to neurons in the RVLM
Horiuchi J, Killinger S, Dampney RA.
Dept. of Physiology and Institute of Biomedical Research, The University of Sydney, New South Wales, Australia. Am J Physiol Regul Integr Comp Physiol. 2004 Dec;287(6):R1335-43. Epub 2004 Jul 22.
In my previous post I have discussed about Ito and Sved's paper which showed that if the CVLM is inhibited first, then blocking the glutamate receptors in the RVLM reduced the arterial pressure. The idea was that RVLM balances its glutamatergic inputs (which have direct excitatory action on the pre-sympathetic neurons) and GABAergic inputs (which have inhibitory action) resulting in no changes in arterial pressure. This idea was challenged by Dampney's group. In this article the authors tested the hypothesis that the inhibition of presympathetic neurons of the RVLM is a balancing act that occurs when excitatory amino acids (EAA) in the RVLM is involved in mediating the excitatory inputs to both the presympathetic neurons and the interneurons in the CVLM.
Dept. of Physiology and Institute of Biomedical Research, The University of Sydney, New South Wales, Australia. Am J Physiol Regul Integr Comp Physiol. 2004 Dec;287(6):R1335-43. Epub 2004 Jul 22.
In my previous post I have discussed about Ito and Sved's paper which showed that if the CVLM is inhibited first, then blocking the glutamate receptors in the RVLM reduced the arterial pressure. The idea was that RVLM balances its glutamatergic inputs (which have direct excitatory action on the pre-sympathetic neurons) and GABAergic inputs (which have inhibitory action) resulting in no changes in arterial pressure. This idea was challenged by Dampney's group. In this article the authors tested the hypothesis that the inhibition of presympathetic neurons of the RVLM is a balancing act that occurs when excitatory amino acids (EAA) in the RVLM is involved in mediating the excitatory inputs to both the presympathetic neurons and the interneurons in the CVLM.
In order to test this hypothesis the authors inhibited the CVLM neurons and measured the effects of EAA receptors blockade on mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA). Similar to the previous study the male SD rats were bilaterally injected with muscimol in the CVLM, which increased MAP and HR. In addition to that the authors also tested the RSNA which is also increased. Subsequently when kynurenic acid was injected in the RVLM it decreased the MAP significantly compared to vehicle injection but there were no changes in the HR and RSNA. After 50 minutes of injection all three parameters returned to the baseline level with MAP just below it and HR and RSNA above it. The results show that inhibition of tonic glutamatergic inputs to the neurons in the RVLM has minimal effect on the activity of RVLM presympathetic neurons. These results were quite contradictory to what was reported previously by Ito and Sved.
Tonic glutamatergic drive of RVLM vasomotor neurons?
Alan F. Sved
Dept. of Neuroscience, Univ. of Pittsburgh, Pennsylvania. Am J Physiol Regul Integr Comp Physiol 287: R1301–R1303, 2004;
Dr. Sved gave an editorial comment on the above article, in which he have reports that the decrease in MAP in the above study was not associated with a decrease in RSNA. He also points out a number of differences between the two studies. The anesthetic usage was different (Ito and Sved used intravenous chloralose or urethane after induction of anesthesia with halothane, where as Horiuchi et al. used intraperitoneal urethane, which could increase the plasma osmolality and may cause an effect on the central neural vasomotor control). The timing and speed of injections are not reported but may play a role in the difference seen between the two experiments. Most importantly the exact sites of injections, even though the center is identical, the angle of the head, pipette or design of pipette tip may produce differences.
Tonic glutamatergic drive of RVLM vasomotor neurons?
Alan F. Sved
Dept. of Neuroscience, Univ. of Pittsburgh, Pennsylvania. Am J Physiol Regul Integr Comp Physiol 287: R1301–R1303, 2004;
Dr. Sved gave an editorial comment on the above article, in which he have reports that the decrease in MAP in the above study was not associated with a decrease in RSNA. He also points out a number of differences between the two studies. The anesthetic usage was different (Ito and Sved used intravenous chloralose or urethane after induction of anesthesia with halothane, where as Horiuchi et al. used intraperitoneal urethane, which could increase the plasma osmolality and may cause an effect on the central neural vasomotor control). The timing and speed of injections are not reported but may play a role in the difference seen between the two experiments. Most importantly the exact sites of injections, even though the center is identical, the angle of the head, pipette or design of pipette tip may produce differences.
C1 neurons in the rat rostral ventrolateral medulla differentially express vesicular monoamine transporter 2 in soma and axonal compartments
C. P. Sevigny, J. Bassi, A. G. Teschemacher, K. S. Kim, D. A. Williams, C. R. Anderson and A. M. Allen
European Journal of Neuroscience, Vol. 28 pp. 1536-1544, 2008
C1 neurons of the RVLM are defined as producing adrenaline, but the effect that adrenaline is used to is still not quite worked out; they are generally accepted as using glutamate as their major neurotransmitter. To this end, they express a vesicular glutamate transmitter. They also express a vesicular adrenaline transporter: VMAT2. The authors of this paper looked at the differential expression of VMAT2 in the cell soma and axon terminals to determine if rostrally-located, bulbospinal C1 neurons release adrenaline somatodendritically (which requires cell body expression of VMAT2), synaptically, or both.
A lentivirus was used to identify the C1 neurons in one group of rats, rhodamine fluorescent microspheres were injected into the T3/T4 level of the spinal cord to identify spinally projecting neurons in a second group of rats, and anti-PMNT and anti-VMAT2 antibodies were used to identify PMNT (again, to identify C1 neurons) and VMAT2 in tissue sections. Both brainstem sections and spinal cord sections were examined, to compare cell soma expression of VMAT2 in spinally projecting C1 nuerons to axonal expression in the same neurons. Additionally, spinally projecting C1 neurons were compared to rostrally projecting C1 neurons.
Rostrally projecting C1 neurons did have VMAT2 present in their cell bodies, but bulbospinal C1 neurons, which were identified by the presence of the rhodamine fluorescent microspheres, rarely did. VMAT2 was present in the axon varicosities in the spinal cord, though, suggesting that the bulbospinal C1 neurons do release adrenaline as a neurotransmitter. While bulbospinal C1 neurons may not release adrenaline somatodendritically, though, some C1 neurons are thought to, and bulbospinal neurons seem to be excited to alpha-2-adrenoceptor blockade, suggesting that adrenaline may have a role in regulating the tonic activity of the RVLM. In short, some C1 neurons, but not the bulbospinal C1 neurons, likely release adrenaline within the RVLM (which has an effect on tonic activity in the RVLM) while C1 bulbospinal neurons release adrenaline along with glutamate at their axon terminals.
Thursday, June 9, 2011
Tonic glutamate-mediated control of rostral ventrolateral medulla and sympathetic vasomotor tone.
Ito S, Sved AF.
Am J Physiol. 1997 Aug;273(2 Pt 2):R487-94.
Previously studies by Guyenet et al., showed that when glutamate receptors are blocked bilaterally in the RVLM using kynurenic acid (glutamate receptor antagonist) it produced no significant changes in the arterial pressure suggesting that glutamate may not play a role in tonic activation of RVLM pre-sympathetic neurons. In this article the authors questioned this notion. The authors hypothesized that if CVLM-mediated inhibition of RVLM is removed then excitatory amino acids (EAA) could provide neuronal excitation and in turn an increase in arterial pressure. Male Sprague-Dawely rats were used for this study. Two different types of anesthesia were used in this experiment, alpha-chloralose or urethane both given intravenously to show that changes seen were not specific to anesthetic that is used. The major finding of the present study is that when CVLM was inhibited by micro injection of muscimol (GABA-A receptor agonist), it markedly increased the arterial pressure, subsequently when kynurenic acid (kyn) was injected into the RVLM it decreased the elevated arterial pressure. Interestingly when only kyn was injected it produced mild changes in the arterial pressure. These findings show that the when CVLM was inhibited it increased arterial pressure and this could be mediated through RVLM neurons excited by EAA. One possible explanation for this results is that on one hand the glutamatergic inputs to the RVLM are excited to activate the sympathoexcitatory neurons and on the other hand inhibited through the CVLM.
Am J Physiol. 1997 Aug;273(2 Pt 2):R487-94.
Previously studies by Guyenet et al., showed that when glutamate receptors are blocked bilaterally in the RVLM using kynurenic acid (glutamate receptor antagonist) it produced no significant changes in the arterial pressure suggesting that glutamate may not play a role in tonic activation of RVLM pre-sympathetic neurons. In this article the authors questioned this notion. The authors hypothesized that if CVLM-mediated inhibition of RVLM is removed then excitatory amino acids (EAA) could provide neuronal excitation and in turn an increase in arterial pressure. Male Sprague-Dawely rats were used for this study. Two different types of anesthesia were used in this experiment, alpha-chloralose or urethane both given intravenously to show that changes seen were not specific to anesthetic that is used. The major finding of the present study is that when CVLM was inhibited by micro injection of muscimol (GABA-A receptor agonist), it markedly increased the arterial pressure, subsequently when kynurenic acid (kyn) was injected into the RVLM it decreased the elevated arterial pressure. Interestingly when only kyn was injected it produced mild changes in the arterial pressure. These findings show that the when CVLM was inhibited it increased arterial pressure and this could be mediated through RVLM neurons excited by EAA. One possible explanation for this results is that on one hand the glutamatergic inputs to the RVLM are excited to activate the sympathoexcitatory neurons and on the other hand inhibited through the CVLM.
Wednesday, June 8, 2011
Subscribe to:
Posts (Atom)