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Hypothalamic Neural Circuits and Immunohistochemistry

Introduction

This report will provide a succinct summary of hypothalamic neural circuits (HNC) and Immunohistochemistry. Specific discussion will address the anatomy and role in HNC as a regulator of sleep-wake transition. Anatomy discussion will center on understanding the role and functionality of components that make up the arousal and sleep systems. This report will also address the use of immunohistochemistry in free-floating brain sections to examine gene expression in the brain.

The neurophysiology of sleep and wakefulness

The study of neurophysiology is critical due to the prevalence of the disorder. In the United States there are approximately fifty to seventy million individuals who deal with chronic sleep disorder. The three impacted areas include compromised daily functions, weakened immune system, and contracted life quality. There is also an interconnectedness between the various cell groups that affect behavioral states. This subsequently creates a need to understand the neural circuitry in order to address the disorder (Schwartz & Roth, 2008).

The arousal system is designed to operate by having cell groups’ fire in predictive patterns. In the ascending cell population there are critical cell groups including cholinergic, noradrenergic, serotoninergic, dopaminergic, and histaminergic neurons. These various cell populations are labeled in groups including PPT, LDT, and TMN (Schwartz & Roth, 2008).

PPT is the Pedunculopontine, the LDT is the laterodorsal tegmental nucleus, and the TMN is the tuberomammillary nucleus. Subsequently there is a systematic and scientific interaction between those arousal cell systems and the VLPO. The VLPT is known as the ventrolateral preoptic nucleus (Schwartz & Roth, 2008).

When there is harmony between the cell groups and sleep-wake systems, the body should operate assiduously. However, sleep disorders represent a pathology that requires detailed examination of the biology of the arousal-sleep system. There are two identified branches in the arousal system. The first branch aptly supplies the thalamus with nerves, this activates relay neurons and reticular nuclei. This successive process is critical for thalamocortical transmission. The PPT/LDT nuclei are triggered by projections coming from cholinergic structures in the brainstem and basal forebrain (Schwartz & Roth, 2008).

Rapid eye movement (REM) and general wakefulness are the periods where the PPT/LDT nuclei are active the most. The second branch projects into the lateral hypothalamus, basal forebrain, and the cerebral cortex. In summation, cholinergic neurons, monoaminergic cell populations, and other neurons within the ascending arousal system discharge. The objective is to behave in a coordinate pattern that provides the necessary cortical arousal (Schwartz & Roth, 2008).

During sleep, the neurons of the VLPO effectually block the circuits that trigger the cortical. Orexin (hypocretin) is a neuropeptide created by the neuronal cluster in the posterior portion of the lateral hypothalamus. According to some theories the interconnectivity between orexin/hypocretin and awakening, and sleep-promoting cell groups are important. Theoretically, they may offer balance and regulation in the sleep-awake system (Schwartz & Roth, 2008).

Sleep Regulation

Although there are many aspects of the sleep-wake disorder that are unknown, various theories persist. For example Borbeley and colleagues as cited in Schwartz & Roth (2008) purport a two-process model of sleep regulation. The homeostatic or equilibrium component of the body may be triggered by a substrate or protein. This substrate may actually cue the body that it needs to sleep, after long periods of being awake. The rhythmic biological cycles (circadian) naturally occurs every 24 hours (Schwartz & Roth, 2008). Another probable factor triggering the need to sleep, is adenosine. When the body is awake it utilizes stored glycogen (the body’s primary energy source). The glycogen when broken down becomes adenosine which when increased triggers the need to sleep. The second phase of the process sleep theoretically would restore glycogen into the body. Subsequently, as glycogen is increased in the body, specifically the forebrain the body can again sustain an awake state (Schwartz & Roth, 2008).

Summary

Various sleep disorders include Alzheimer’s, Parkinson’s disease, Insomnia, and Narcolepsy. The treatment of relevant conditions often requires a combination of behavioral and pharmacological therapies. Suggested behavioral approaches include cognitive behavioral, and sleep-restriction therapy. Other approaches include the use of hypnotic agents and sympathomimetic alerting agents. Also, primary sleep-wake disorders have been treated using (CPAP) continuous positive airway pressure, antidepressants and sodium oxybate for narcolepsy.

Finally, there are identified physiological patterns of sleep and wakefulness that have been briefly identified. Various brain-processing networks and neurochemical systems are affected differently. Continuous research will further understand the nature, and functioning of causative relationships between the sleep-awake states. It is noteworthy that there is no customized approach for treating any individual. What is important is that any proposed treatment is carefully regulated. This is because patients who have a history of substance abuse may experience complications as a result of the proposed medication. Presently amphetamines are a wake-promoting drug that can lead to undesirable abuse. Carefully monitored it may provide treatment for disorders like narcolepsy.

Immunohistochemistry

Background and Explanation

Immunohistochemistry is known as (IHC). IHC is a method that identifies the existence and the location in the body of proteins. These proteins are in various tissue sections. IHC is not as sensitive as Western blotting or ELISA. However, the value is that it allows for process observation with the presence of intact tissue. This is useful for example when studying the progression and treatment of chronic diseases. The use of IHC does not discourage or discredit the use of other methods. In fact IHC when combined with microscopy offers a macro perspective of data obtained from other processes (ABCAM, n.d.).

In order to identify the target protein researchers stain the tissue section with antibodies. These antibodies effectively recognize the target protein. The antibodies are designed to only bind with targeted protein in the tissue section. To view the antibody-antigen interface the use of chromogenic detection (CD) or fluorescent detection (FD) are used. With CD an enzyme conjugated to the antibody attaches with a substrate to create a colored precipitate at the protein source. In contrast FD enables the researcher to view the flurophore (which is conjugated to the antibody). FD is viewed by using fluorescence microscopy (ABCAM, n.d.).

Application 1

The DNA repair protein (MGMT) O6-Methylguanine-DNA methyltransferase presently indicates resistance to alkylating agents. Therefore two approaches MSP (methylation-specific polymerase chain reaction), and (IHC) immunohistochemistry are used to detect MGMT protein expression. This report did not indicate which method IHC or MSP was more effective, at detecting MGMT protein expression (Brell, Ibanez, & Tortosa, 2011).

Specific methodology included an examination of patients with brain tumors and those with other forms of cancer. In order to be included in the study both studies had to be used as a diagnostic test for detecting MGMT protein expression. For this meta-analysis study, results indicate that IHC does not provide similar results as the MSP test when assessing MGMT protein expression. This study reveals several important facts. The first fact is that brain tumors show the highest degree of discordance between MSP and IHC results (Brell, Ibanez, & Tortosa, 2011).

Application 2

Another study sought to determine whether or not CB2 receptors are expressed on neurons in the brain. Some studies have indicated that CB2 receptors are not noticeable on neurons with normative circumstances, while other studies have revealed the opposite. This report sought to reveal causative factors such discrepancies. Those include dependence on not fully validated CB2 receptor antibodies and IHC procedures. Results indicate that the CB2 antibodies tested are not useful for studying CB2 expression in the brain (Baek, Darlington, Smith, & Ashton, 2013)

Researchers have expressed concern about the efficacy of CB2 receptor antibodies to localize CB2 receptor expression in the brain. There have been a number of previous tests including studies by Gong et al. (2006) and Suarez et al., (2008 & 2009), cited by Baek, Darlington, Smith, & Ashton, (2013). Suarez and his contemporaries’ performed IHC studies which revealed prevalent brain immunolabeling. However, in the Suarez et al., study the non-commercial antibody was not duplicated in this study.

According to Rhodes and Trimmer (2006) as cited by Baek, Darlington, Smith, & Ashton, (2013), there are limitations revealed. Specifically, the use of antibodies to conduct IHC on brain tissue sections was shown to be less effective than other biochemical methods. This is primarily attributed to the complex heterogeneous cellular and molecular construction of the brain. At least compared to other tissues within the body (Baek, Darlington, Smith, & Ashton, 2013).

Tau immunohistochemistry in acute brain injury

One study examined the neuropathological mechanism associated with head injury as a risk factor for Alzheimer’s disease. Also Neuronal cytoskeletal modifications in the form of neurofibrillary tangles and neuropil threads have been identified in young men with head injuries. In fact, these repetitive injuries have resulted in many deaths, during the 20’s. Previous studies have identified an accrual of tau within neuronal somata and damaged axons (Smith, Graham, Murray, & Nicoll, 2003).

The report hypothesized that patients suffering from a single acute blunt head injury could have tau-immunoreactive tangles in their brain. A total of 45 cases were immunostained in an effort to detect the existence of tau. The groups were divided according to age and survival time. They were also compared to age-match controls. The results revealed that following traumatic brain injury (TBI) there were not more cases of neurofibrillary tangles comparative to age-match controls (Smith, Graham, Murray, & Nicoll, 2003).

Value and limits of immunohistochemistry

Another study investigated systematically diagnostic value and limits of immunohistochemistry using representative tumor specimens of (OG’s) oligodendroglioma’s, (CCEs) clear cell ependymoma’s, and (CN’s) central neurocytoma’s. Data results revealed that (IHC) immunohistochemistry utilizing anti-nuclear protein, anti-vimentin, and anti-epithelial membrane antigen on specific tumor specimens provides identifiable dissimilarities (Koperek, et al., 2004). The identifiable dissimilarities are between central neurocytoma’s versus oligodendroglioma’s, and clear cell ependymoma’s. However, it is noteworthy that because of focal expression of glial proteins of central neurocytoma’s and neuronal proteins in oligodendroglioma’s and clear cell ependymoma’s, IHC has minimal value on small tumor specimens (Koperek, et al., 2004).

Conclusion

The human anatomy is a complex living and breathing entity. Behavioral and cognitive maladies can cause chronic disorders. This means that science and medicine must continually explore the described brain-processing networks and neurochemical systems, to understand existent physiological patterns. This diligent approach will enable humanity to aptly address chronic issues like sleep disorders, Alzheimer’s, and narcolepsy. The use of IHC as an effective method to identify proteins on bodily tissues remains flawed. However, a diligent continuance of such studies will provide viable options for future scientific research and medical advancement.

References

ABCAM. (n.d.). IHC-PARAFFIN PROTOCOL (IHC-P). Retrieved from http://www.abcam.com/ps/pdf/protocols/ihc_p.pdf

Baek, J., Darlington, C. L., Smith, P. F., & Ashton, J. C. (2013, June). Antibody testing for brain immunohistochemistry: Brain immunolabeling for the cannabinoid CB2 receptor. Journal of Neuroscience Methods, 216(2), 87-95.

Brell, M., Ibanez, J., & Tortosa, A. (2011). 06-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction. BioMed Central, 11(1), 35-48.

Koperek, O., Gelpi, E., Birner, P., Haberler, C., Budka, H., & Hainfellner, J. (2004, July). Value and limits of immunohistochemistry in differential diagnosis of clear cell primary brain tumors. Acta Neuropathol, 108(1), 24-30.

Schwartz, J. R., & Roth, T. (2008, December). Neurophysiology of Sleep and Wakefulness: Basic Science and Clinical. Curr Neuropharmacol, 6(4), 367-378.

Smith, C., Graham, D. I., Murray, L. S., & Nicoll, J. A. (2003). Tau immunohistochemistry in acute brain injury. Neuropathology and Applied Neurobiology, 29(1), 496-502.

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