EFFECT OF PRE-NATAL INTERMITTENT MONOTONOUS AND RHYTHMIC AUDITORY EXPOSURES ON BRAINSTEM NUCLEI PLASTICITY OF ONE DAY-OLD CHICK

Environmental auditory exposure in a form of both enrichment and stressor environmental can differently modulate the development of auditory system Prenatal chronic noise exposure caused neurogenesis and neuroplasticity disorders in the brain's auditory pathway resulting in neurocognitive impairment. Meanwhile exposure to prenatal music positively modifies morphological and biochemical developments in the brain's auditory pathway that supports neurogenesis and neuroplasticity. This study aimed to determine the plasticity of brainstem nuclei after exposure to music, noise, and a combination of both. This research used an experimental method using 24 female chicks as subjects. Subjects were obtained from eggs after incubation, which were divided into 4 groups: control, music, noise, and combined noise and music. Brain stem nuclear plasticity was measured by total number of nuclei, neuronal nuclear area, and synaptophysin expression as parameters. Prenatal music exposure significantly increased the total number of neurons, neuronal nuclear area, and synaptophysin expression in brain stem nuclei (p < 0.001), whereas combined and noise exposure significantly decreased these three plasticity parameters (p < 0.001). In conclusion, prenatal music exposure potential to increase neuroplasticity of brainstem nuclei for better neurocognitive function


BACKGROUND
Pre-natal period is a sensitive period since environmental stimuli interact with genes in influencing the formation and refinement of neural connectivity of the developing brain (Linden et al., 2003).Auditory stimulation in a form of both environmental enrichment, such as music, and stressor, such as noise, can differently modulate the development of auditory system and hippocampus.
Both auditory system and hippocampus as its higher order integration of sensory input have direct and indirect connections (Kirste et al, 2015).Decreased expression gephyrin that alter inhibitory postsynaptic complex, receptors clustering of synaptic (Kumar et al., 2014).Synapses are thus of primary importance in understanding the basis of such improved behavioral and morphological correlates.Increased expression of synaptic proteins also reflects synaptic strength, which depends upon target contact and synaptic activity (Garner et al., 2002).one of important synaptic protein is synaptophysin.It is a major integral glycoprotein membrane protein which has weight 38-kDa.Expression of synaptophysin occurs prior as well as parallel to the formation of synapses, and is considered as a marker of synaptogenesis (Plunkett et al., 1998).In the present study, the effect of prenatal combined chronic auditory exposure expression of synaptic proteins in the chick brainstem auditory nuclei nucleus magnocellularis (NM) and nucleus laminaris (NL) during development and at hatching was assessed.
In mammals, fetus may directly elicit stress response to the external stressor.Furthermore, mother's stress hormones may pass through the placenta, followed by placental stress response (Canlon et al., 2003).In order to focus on examining the impact of pre-natal chronic auditory  (Kauser et al., 2011;Alladi a et al., 2005).As an embryo that independent, fertilized egg of chicken provide an excellent system to study the effects of prenatal acoustic exposure.

Subject and Incubation
Isa Brown domestic chicks (Gallus gallus domesticus) were used as the experimental model.from the embryonic day 10 (E10) until hatching.

Experimental Group
The protocol and number of animals used in from the embryonic day 10 (E10) until hatching.
Except combined group will get 15 minutes noise and 15 minutes music with 15 minutes respectively.

Tissue collecting and Processing
The

RESULT The Total Number of Neurons
The total number of neurons (mean±SD) of the individual brainstem auditory nuclei, nucleus magnocellularis

Immunoreactivity
The difference in the staining intensity between the sections among the group was assessed using Image J with IHC profiler plug in.
Image analysis of immunostaining sections of control embryos, in both NM as well as NL, showed an increase in synaptophysin immunoreactivity in music group.statistical analysis with one way ANOVA showed significant difference (p< 0.001).
and significant decrease in noise group (p< 0.001).
There was no significant difference between control group and combined group (p= 137).Figure 3 (Meyer, 1983).Prenatal auditory exposure using music has been reported increased synthesis synaptophysin and syntaxin 1 in chick brainstem auditory nuclei.This condition beneficially enhances positive effect to develop auditory system (Alladi a et al., 2002).In this study we found that there was a significant difference between the mean optical density among the group that immune-positive to synaptophysin.Music group has the highest optical density while the noise group has the smallest optical density.There was a significant difference in the combined group with the music and noise groups but there was no significant difference with the control group.This is in accordance with previous studies of auditory exposure to music increases the value of optical density against synaptophysin and vice versa noise exposure could decrease optical density in different parts of the brain.While optical density in the combined exposure group showed improvement.That may be due to exposure to music after exposure to noise because there is a significant difference with the noise group.This suggests that exposure to music increases the level of synaptophysin associated with an increase in the number of synaptic vesicles as well as the formation of a new axon terminal (Chaudhury and Wadhwa, 2009;Chaudhury et al., 2009).

CONCLUSION
In conclusion, prenatal exposure to music improve the neuroplasticity of brainstem nuclei of the chicks.Neuroplasticity in brainstem is very important because it is the auditory pathway that delivers afferent stimuli to another region in the brain.Although could not prevent neuron cell apoptosis in early development.

Fertilized
eggs day 0 of healthy chicks, weighing 50-60 g, were obtained from PT Sierad Tbk., and incubated in a double insulated egg incubator.Temperature controlled incubator of 37 ± 1° C and humidity 70% ± 2% was used for incubating the eggs.Tilting of eggs was automatically controlled for 4 times a day and a photoperiodicity of 12:12 h day and night cycle was maintained in 21 days incubation period.The incubator has a forced draft of air for aeration.On day 9.5 of incubation to make access to sound, a part of the eggshell removed keeping the membranes intact at the animal pole over the air sac approximately 2-5 mm size.The fertilized eggs were given prenatal music stimuli through speakers built in the incubator.The sound exposures were for 15 minutes per hour, over a period of 24 hours (total 6 hours per day) chicks post hacth 1 from all of group were anesthetized with anesthetic ketamine 40 mg/kg weight and sacrificed by decapitation.Brains were dissected out, the brainstem cut and fixed in 10% normal buffer formalin for 3 days.The next step was dehydration into stratified alcohol to allow the water in the tissues to be removed and using xelene to clearing.Then the tissue is inserted into the liquid paraplast and in the final embeded in to paraffin blocks.Paraffin blocks cut coronal sections of 5 µm thickness.The section stained by cresyl violet and immunohistochemistry to localize the synaptic proteins synaptophysin.

Figure 3 .
Figure 3. Immunohistochemistry staining showed immunoreactivity to synaptophysin 40x of NM.A-D control, music, noise, combined.The music group has the highest synaptophysin immunoreactivity.

Figure 4 .
Figure 4. Immunohistochemistry staining showed immunoreactivity to synaptophysin 40x of NL.A-D control, music, noise, combined.The music group has the highest synaptophysin immunoreactivity.

Figure 5 .
Figure 5. Histogram optical density immunohistochemistry of synaptophysin on both brainstem nuclei.

Table 1 .
Data of All Group Showing Neuron Number and Neuronal Nuclear Area (µm 2 ) : One way ANOVA of both neuron number among and mean neuronal nuclear area of four groups were same, p< 0,001. Notes