Manganese-enhanced MRI to evaluate neurodegenerative changes in a rat model of kainic acid-induced excitotoxicity

Authors: Melda Apaydin...Dilek Taskiran

Background: Kainic acid is a neuro-excitotoxic agent that can be used to study neurodegeneration in animals such as rats. As a specific agonist to kainate receptors kainic acid is able to modulate transmission and excitability. For this study, MeMRI was used to study in-vivo changes of neurons in response to kainic acid-induced excitotoxicity.

Methods:

• KA unilateral injections
• MeMRI (I.P 40mg/kg one injection)
• Bax immunohistochemistry
• Timm's Staining

Results:

• Hyperintensity was observed, as expected, in the hippocampal area in the side of the KA-injections. With a significant difference found between the ipsilateral sites of the injection and the contralateral sites.
• Bax immunohistochemistry was used to assess apoptotic cell death in regions induced with KA, and revealed a significant increase in the KA treated regions compared to the saline treated regions.
• The Timms staining showed increased mossy fiber sprouting and axonal plasticity in CA1 and CA3 granule cells in the hippocampus in KA treated regions compared to the control regions.

~JI

Recording sympathetic nerve activity chronically in rats: surgery techniques, assessment of nerve activity, and quantification

Stocker, S.D. and Muntzel, M.S. Recording sympathetic nerve activity chronically in rats: surgery techniques,assessment of nerve activity, and quantification. Am J Physiol Heart Circ Physiol 305: H1407–H1416, 2013. This review article covers the latest advances in recording chronically from sympathetic nerves focussing on recordings in rats. The article goes in significant details on the methods and provides some nice diagrams for recording from lumbar, renal, and splanchnic nerves. It also has a very good section on "Distinguishing Between Sympathetic Nerve Bursts, Background Noise, and Electrical Artifacts." This would be a good primer for anyone who is just starting to recording from sympathetic nerves or wants a refresher on what to look for in determining very good, versus good, versus bad sympathetic nerve recordings. Another section entitled "Time Course and Reasonable Expectations of Chronic SNA Recordings in Rats" covers topics like the Postimplant recovery period where sympathetic activity tends to be high because of a number of stress-related effects of surgery. The last section covers "Analysis of SNA Signals" and breaks down the signals into components like frequency of bursts versus amplitude of bursts. Since we record all of our raw nerve activity on the Powerlab, this could be a useful way to do some data mining and look for specific changes in sympathetic nerve activity betweeen active and sedentary animals that we might not have seen simply comparing voltages between groups. All-in-all this is a very useful review and I would consider required reading for anyone involved in experiments examining sympathetic nerve recordings, chronic or acutely. ~PJM

NTS A2a adenosine receptors inhibit cardiopulmonary chemoreflex of sympathetic outputs

Zeljka Minic, Donald O'Leary, Tadeusz Scislo

Background:
The nucleus of the solitary tract is known to be a primary modulator of the cardiopulmonary chemoreflex (CCR) via adenosine receptors (A1 and A2a). Unlike the rest of the CNS, the A2a is the primary receptor within the NTS compared to the A1 receptors.  Stimulation of the A2a receptors in the NTS shows regional specific changes in sympathetic nerve activity output such as decreases in RSNA or increases in pre-ASNA.  Here the question was whether stimulation of the A2a receptors in the NTS would promote region specific inhibition of the CCR.

Methods:

• Microinjections into the NTS
• Neuron recording

Results:
Although it is seen that stimulation of A2a receptor causes differential effects on SNA, it was shown in this study that stimulation of A2a receptor causes universal inhibition of CCR responses. Presenting for the first time, that A1 and A2a receptors my function synergistically to control cardiovascular reflexes through different mechanisms.

~JI

Tonic Glutamatergic Input in the Rostral Ventrolateral Medulla Is Increased in Rats With Chronic Heart Failure

Tonic Glutamatergic Input in the Rostral Ventrolateral Medulla Is Increased in Rats With Chronic Heart Failure by Wei-Zhong Wang, Lie Gao, Han-Jun Wang, Irving H. Zucker, Wei Wang This is a paper from the Nebraska group in which they looked at tonic excitation of the RVLM in chronic heart failure (CHF) rats. Microinjections of kynurenic acid (NMDA and AMPA receptor antagonist), AP5 (NMDA receptor antagonist), or CNQX (AMPA receptor antagonist) into the RVLM of CHF but not sham rats each reduced resting blood pressure and renal sympathetic nerve activity (RSNA). In addition direct recordings from single unit bulbospinal RVLM neurons revealed that kynurenic acid decreased the already elevated firing frequency of RVLM units from CHF but had not sham rats. Sham rats were those that had had the open chest surgery required for the coronary ligation CHF model without actually tying off the coronary artery. Sham and coronary ligated animals were studied 6-8 weeks after the ligation or sham surgery. The conclusions of the paper are that glutamate activing via both NMDA and non-NMDA (likely AMPA) receptors located on RVLM neurons contributes to elevations in resting RVLM neuronal firing and increased basal SNA in heart failure. Because antagonists were used it was not possible to determine whether the increase glutamate neurotransmission was due to increased input or a change in post-synaptic receptors or transduction mechanisms. Because unit recordings confirmed a glutamate senstive increase in resting firing frequency these data suggests that elevated SNA was due at least in part to increased firing of RVLM neurons rather than downstream changes at the level of the spinal cord or sympathetic ganglion. A prime region that could contribute to increase glutamatergic input is the paraventricular nucleus of the hypothalamus, a brain regions that has been extensively studied by this group in previous studies related to heart failure. Finally, the PVN is known to be overactive in CHF animals and it also has direct glutamatergic projections to the RVLM so it's logical to expect it is this connection that serves to drive excessive SNA in CHF. ~PJM

Diet-induced obesity severely impairs myelinated aortic baroreceptor reflex responses

Belinda H. McCully, Virginia L. Brooks and Michael C. Andresen

Obesity leads to impaired baroreceptor reflex responses. This article investigated whether the baroreflex input to the brainstem about  around resting or non resting bp was altered. They basically stimulated the aortic depressor nerve (ADN) on one side after removal of the carotid nerve inputs to the brainstem was removed. All animal were put on a high fat diet and those that became obese were put into the obese prone group and those that did not meet the criteria for obesity were put into the obesity resistant group. If the animal were somewhere in between then the animals were thrown out.They then did used different voltages to stimulate the ADN  in order to to see if there were differences between control, obese resistant and obese prone animal. The data showed that the OP had attenuated  response to stimulation of the ADN at lower volts compared to the other two groups. 

Conclusion: This data suggests that Obesity lead to to impaired baroreceptor reflex control of resting blood pressure.

-MD

Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells.

Authors: Young-Goo Han..Arturo Alvarez-Buylla

Background: Granule neurons in the hippocampal denate gyrus, more specifically the subgranular zone (SGZ), receive information from the cortex and higher brain regions and participates in learning and memory formation.  During development these granule neurons are developed from granule neuron precursors (GNP) that continue to develop substantially directly after the birth and less rapid throughout the rest of a lifetime.  Recently, it has been found that the cilia on cells plays a substantial role during development, by concentrating receptors and signal transducers required for growth using the intraflagellar transport machinery (IFT).  This article shows how cilia along with Shh signaling are essential for establishment and expansion of GNP's in the postnatal denate gyrus.

Methods:

• cell counting
• in-situ hybridization
• qPCR

Results:

• Cilia null mice (Kif3a) were found to have normal morphology of the Ammon's horn on the hippocampus, however the denate gyruses were much smaller and more disorganized when compared to the wild-type mice. GNP's in the mutant mice were also found to be disorganized and reduced, with only 1/3 as many as the wildtype located in the granual layer instead of the SGZ. To verify the results were in fact from the loss of primary cilia, another mutation was induced to mice that inhibited Ift88, a gene encoding for essential subunits of IFT particles.  As shown in previously in Kif3a, Ift88 mice 1/3 of the primary cilia was disfuctional leading to severe decrease in GNP proliferation. Finally it was shown that primary cilia are responsible for properly functioning transcription signal pre and post birth such as Shh, Smo, and SmoM2.  Concluding, that primary cilia in the developing and postnatal nervous system are critical in correspondence of Shh, Smo, and SmoM2 signalling in establishing and expanding granule nuclear precursor cells that allow for the proper growth of the hippocampal denate gyrus.

      ~JI  

Monoclonal Antibodies Defining Distinctive Human T Cell Surface Antigens

Authors: Patrick C. Kung...Stuart F. Schlossman

Background: Before 1979 monoclonal antibodies were produced using heteroantiserums that randomly discovered a correct matching antibody to the specific antigen.  This was seen through rosetting, a process in which antibodies form a flower pattern around a matching antigen present on a erythrocyte. Unfortunately, even when a correct antibody was found, only low titers could be produced, making them clinically irreverent. In this paper, a new technique is introduced that greatly improves the production of monoclonal antibodies, specifically looking at monoclonal antibodies for human T-cell surface antigens.

Methods:

• The hybridoma technique
• Immunofluorescence and Flow Cytometry

Results

• In order to create monoclonal antibodies, myeloma is fused with mouse spleen cells that contain the antigen of interest.  At this time antibody producing plasma cells will bind with cancer plasma cells, and the rest of the plasma and cancer cells will dying.  The remaining fusion cells are then cultured and capable of producing high titers of the specific monoclonal antibody of interest.
• To distinguish which antibodies produced bound to the correct T-cell of interest, fluorescent flow cytometry was utilized.  Multiple T-cell types were introduced to the antibodies of interest with the assumption the antibody would bind to one T-cell type.  This culture was then introduced to fluorescence that attached only to bound antibodies.  Following, the culture was run through a flow cytometer and the fluorescent cells were characterized to evaluate antibody specificity to human T-cells.  After this was done three different monoclonal antibodies were recognized to have specific binding to unique human T-cells. 

        ~JI