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Experimental Design

Question:

Discuss about the Experiment Design for Behavioural Brain Research.

The management of Parkinson’s illness people with a dopamine precursor  like the L-dopa boosts the growth of severe changes in motor response as well as involuntary movements referred to as L-dopa induced dyskinesia, a serious complication of L-dopa therapy associated with Parkinson’s ailment. The true nature of such manifestations can result to experiments concerning the abnormalities of neural function especially in the corticobasal ganglia motor circuit. However, since the modern theory regarding the arrangement of basal ganglia pathways suggests that both the basal ganglia plus the frontal cortex are put in segregated circuits that are parallel, there is a tremendous proof showing the consistence for associative, limbic and sensorimotor domains (Le Jeune et al., 2008).

Ideally, research shows that the motor components of a sub thalamic nucleus as well as the dorso lateral tip have a usual terminal activity at the chorea which is of signifance in dystonia. On the other hand, the metabolic activity of the limbic and associative sub thalamic nucleus is impaired in both scenarios. However, the result initially showed that there exists a pathophysiological disparity between dystonia and the L-dopa induced chorea. According to Le Jeune et al., (2008), it can now be hypothesized that can also reflect the association of opposite pathways in a lopa induced dyskinesia hence increasing the likelihood that it as well relates to the unusual processing of the either associative or limbic information.

Hence the rationale of this paper is to design an experiment that identifies if or not the modulation of a loop in the basal ganglia can alter the output in other loops in the systematic fashion. The write up also looks at the changes in the 2DG accumulation in associative, sensorimotor plus the limbic domains of the basal ganglia and thalamic nuclei of groups that are not primate.

Nineteen female monkeys were put in separate primate rooms under controlled conditions of light, temperature and humidity. Both food as well as water was provided and animal care was being ensured by a registered veterinarian. The experiment was conducted in respect to the European community’s council directive of September for proper care of laboratory animals.

For the experiment protocol, the demographic property showed that every methyl phenyl treated animals monkeys showed a comparable phase of lesion as well as different behaviours. Both the control plus the methyl phenyl treated animals corresponds to those animals in the D25 and D0 groups while dsykinetic plus the nondyskinetic species originated from  a population of those animals validated earlier. For the later animals, they were created administration of L-dopa twice a day for up to eight months at a modified dose made to give complete reversal of Parkinson’s condition. The animals that contracted serious and reproducible dyskinesia were five while four did not.

Results

Regarding the behavioural evaluation, the parkinsonian condition was determined on an animal rating scale using video tape recordings of the animals. After the evaluated a score of zero represented a normal monkey while the score of six and above represented the parkinsonian animal. However, the degree of dyskinesia was measured by use of a dyskinesia disability scale abbreviated as DDS. Zero meant dyskinesia was absent; one meant the condition was mild with rare dyskinetic movements as well as postures. A scale of two mean moderate with outstanding unusual movements and not interfering greatly with the usual behaviour. Three marked regular and sometimes continuous dyskinesia entering via the normal repertoire of activity. A scale of four represented severe continuous dyskinetic activity rendering the animal disabled as well as replacing the actual behaviour (Kirch et al., 2013).

During the event the monkeys were killed, they were injected with 2DG deoxy glucose which was sterile saline. After almost one hour, all the monkeys were killed by a pentobarbital overdose , while the L-dopa treated monkeys got their modified dose of L-dopa a quarter an hour prior to the introduction of 2DG. Their brains were removed and stored in a chemical substance to freeze. The inner tissues were cut into pieces of 20 micrometres where they were thaw mounted onto slides containing gelatine substance. After they were freeze dried, the auto radiographic methyl methacrylate standards plus the serial sections were put to hpyerfilm for sixty days at a temperature of 30 degrees Celsius. After two months, densitometry analysis of autoradiograph was done by means of an image analysis method. Each animal was analysed according to four sections per nucleus by an examiner in respect to the radioactivity levels found in contrast with the standards. Later on, the SEM plus the mean bound radioactivity were subsequently found for every group. 

For different comparisons of information, a one-way ANOVA table was employed to check for the general significance followed by a t test for different comparisons. For the behavioural assessment of different comparisons, the kruskal Wallis non parametric test was employed to test the entire significance followed by a t test corrected by different comparisons via Dunn’s technique. The data were normally distributed plus the significance level of the t test attuned for inequality of measures dispersion like variance if appropriate. Lastly, the analysis was completed using a strata program where a p< 0.05 was taken to be significant.

Discussion

The 2 deoxy glucose uptake was measured with associative, limbic plus sensorimotor domains of basal ganglia as well as in other target groups of such domains. In that aspect, the outer part of the globus pallidus was classified into three territories namely the associative, sensorimotor and limbic as mentioned earlier. The sub thalamic nucleus was split into associative-limbic and sensorimotor domains. The accumulation of the 2DG was calculated as a whole in the inner parts of globus pallidus as well as in the substantial migra reticulate. There are two outputs of the ganglia due to the continuous overlap between the domains in the nuclei. Ideally, three nuclei were observed in the thalamus. Both the ventral lateral nuclei plus the ventral anterior are known to be thalamic targets of motor loop. The ventral anterior was measured on tiny sections of four millimetres anterior to the anterior commisura where the sub thalamic nucleus is absent and the central lateral was measured according to sections of six millimetres to anterior commisura where the sub thalamic nucleus is present. However, a mediodorsal nucleus is referred to as the thalamic output of the associative loop of the BG commonly known as basal ganglia. However, the nucleus of the terminus of the stria was considered to be the limbic output of the basal ganglia.

Advantages of the Designed Experiment

  • The experiment design is simple and can be applied in different disciplines.
  • The research designs are repeatable hence results can be rechecked as well as verified.
  • The researcher can tailor the experiment as well as maintain validity of the design 

Limitations of the Experiment Design

  • It was not possible to control the extraneous variable
  • The results obtained in the experiment may not be generalised to a larger population.
  • The reaction of the test subjects cannot be true indicators.
  • Human errors can also play a key role in validity of the subjects.
  • The reaction of the test subjects cannot be true indicators of behaviour in non-experimental environments.

In the whole domains of the globus pallidus, 2 deoxy glucose was relatively higher in parkisonian monkeys than the normal monkeys. Both the dyskinetic as well as nondsykinetic animals showed normalised levels of 2 deoxy glucose accumulation in the globus pallidus sensorimotor and limbic. Also, those dyskinetic showed a reduced 2 deoxy glucose uptake in the globus pallidus associative contrary to the methyl phenyl treated as well as non dsykinetic organisms. The 2 deoxy glucose in the two domains were defined in the ST nucleus. Those animals treated with methyl phenyl showed a tremendous decline in 2 deoxy glucose accumulation in the limbic and sensiromotor domains. However, the L- dopa treatments led to a raise in the 2 deoxy glucose levels in the nondsykinetic as well as the nondsykinetic groups. Also, there was no significance group noted within the control groups.

The afore mentioned results shows that while nondsykinetic animals presented normalised metabolic activity compared to the control groups dsykinetic organism were distinguished by a tremendous shifts in the 2 deoxy glucose  accumulation in associative plus limbic linked structures but not by sensorimotor nuclei (Kirch et al., 2013). The results were obtained from all the animals and those that had died half an hour following L-dopa administration. During this period the D5 parkinsonian symptoms were developed and none of the LID was noted. Instead, LID erupted in the 45th minute onwards in the dsykinetic group and since data obtained from one group is not enough for statistical analysis, a qualitative analysis would provide more insights. Following half an hour after dopamine was administered, 2 deoxy glucose levels in motor nuclei were similar to the nondsykinetic and dsykinetic organisms at an hour after L-dopa was administered.

Advantages of the Designed Experiment

The primary finding of this experiment is that neural mechanism accountable for LID expressions may too involve associative plus limbic domains but not the sensorimotor domain. Besides, qualitative temporal analysis supports such aspect since in the dsykinetic organism killed when parkiansonian motor abnormalities were developed and prior to the appearance of LID (Kravitz et al., 2010). Therefore, it can be argued that dsykinesia is associated with the pathological metabolic movement in the associative limbic nuclei. The later should not be considered as a movement disorder but also as entailing motivational , affective as well as cognitive aspects of an individual behaviour which been investigated partially in the clinic.

The pattern of changes of 2 deoxyglucose accumulation in this experiment matches the original findings from other scholars. For instance, it can be conformed the differential metabolic activity between the components of the ST nucleus and the dorso lateral tip in dsykinetic and nondsykinetic organisms (Sebastianutto et al., 2017). Also, the metabolic activity of basal ganglia motor related structure was not affected in any way in the dsykinetic organism compared to the nondsykinetic. However increased 2 deoxy glucose intake in the GPI must conform to the increased GABAergic tone emanating from the striatum and also from the GPe. According to Kravitz et al., (2010), the reduced 2 deoxy glucose accumulation in the MD of dsykinetic organisms would therefore be the signature impairment of the whole circuit. Since the present study only presents a contaminant variation, it does not at any given moment create a causative connection that remains to be demonstrated directly.

Since basal ganglia pathways are considered to be arranged parallel in segregated circuits there is proof showing the continuous funnelling information between the limbic, associative and sensorimotor domains. The same concept was further developed by different scholars by postulating that the same information is continuously by diverse functional domains of basal ganglia via the certain pathways of functional interaction via the basal ganglia circuits. This hypothesis has received lots of accolade from the demonstration that cocaine self-administration produces a continuous involvement sensorimotor, limbic and associative domains. However, chronic L-dopa treatment as well as cocaine administration lead to raised dopamine levels within the striatum can lead to continuous dissemination of information rather than focused processing (Kirch et al., 2013). However, in this write up evidence for comparable mechanisms is being provided but not in the same order that is sensorimotor, followed by limbic land lastly is associative domain.

Together with clinical observations reporting reward deficiency syndrome or the learning deficits in the L-dopa treated persons, it can be postulated that dsykinesia needs no to be seen as a movement disorder, but also as a motivational, affective as well as cognitive disorder. Since the qualitative temporary analysis shows that causative duty of involvement of limbic plus associative nuclei and the direct electrophysiological are mandatory when it comes to the establishment of casual; relationships. Ideally, a shift in the 2 deoxy glucose uptake does not show alteration in firing activity of neurons but shows the integration of shifts during the 45th minute in the entire phase of intrinsic synaptic activity. Moreover, a significant result of this experiment is that LID cannot be continued to being analysed via investigating the motor areas thus rendering unreliable each and every other study that does not pay close attention to the functional organisation of the ganglia loops (Le Jeune et al., 2008). Viewing LID is caused as a result of unwanted involvement of limbic as well as associative aspects or as having the or by having limbic and cognitive abnormalities counterparts of LID as is regularly reported by the hyperkinetic complications. For example, say the electrophysiological investigations conforms to the causative hypothesis , modulating the activity of non-motor areas would drastically lower severity of LID , hence offering new drug targets for treatment of the same condition.

References

Le Jeune, F., Peron, J., Biseul, I., Fournier, S., Sauleau, P., Drapier, S., ... & Herry, J. Y. (2008). Subthalamic nucleus stimulation affects orbitofrontal cortex in facial emotion recognition: a PET study. Brain, 131(6), 1599-1608.

Kirch, R. D., Meyer, P. T., Geisler, S., Braun, F., Gehrig, S., Langen, K. J., ... & Döbrössy, M. D. (2013). Early deficits in declarative and procedural memory dependent behavioral function in a transgenic rat model of Huntington's disease. Behavioural brain research, 239, 15-26.

Kravitz, A. V., Freeze, B. S., Parker, P. R., Kay, K., Thwin, M. T., Deisseroth, K., & Kreitzer, A. C. (2010). Regulation of parkinsonian motor behaviours by optogenetic control of basal ganglia circuitry. Nature, 466(7306), 622-626. 

Sebastianutto, I., Cenci, M. A., & Fieblinger, T. (2017). Alterations of striatal indirect pathway neurons precede motor deficits in two mouse models of Huntington's disease. Neurobiology of Disease.

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