UB201L

Ankur Bhaumik
18895

Neurobiology Assignment Lab 1 and 2

Upper word limit: 200 +/- 20 (2.5 marks each)

Q1. In A clinical trial of a drug X, the motor deficit was observed in mice and was suspected
that it could be a result of myelination loss. Design an experiment to investigate the reason(s)
for the observed motor deficit.
➢ We observe the distribution myelin in the two cases i.e, with the administration of the
drug to a mouse and without it (control).
Black Gold II stain can be used. It is specifically used in visualization of Myelin in the
Central nervous system. Large myelin tracts are stained dark red-brown and single
myelin fibers are stained black.

The following procedure is followed-

1. Profusion of both mice with 4% PFA and processing in 30% sucrose.
2. Brain dissection.
3. Brain sectioning using cryostat at -18deg.C to -20 deg.C
4. Staining with Black Gold II
5. Post staining processes like expulsion of excess stain by sodium thiosulphate,
6. Clearing and processing with mounting media.
7. Mounting and observation.

If the motor deficit is due to myelination loss, we are likely to observe-

1. Much less intense staining in the mouse which was administered the drug than
the control. This suggests overall myelination loss.
2. Thinner myelin tracts in the mouse which was administered the drug than the
control.
3. More regions stained black and lesser regions stained dark red brown. This
would suggest degeneration of myelin tracts into fibres.

Q2. A fluorophore is known to have high affinity for the specific protein “X” from a western
blotting experiment. It is suspected that trace amounts of this protein “X” is present in the
brain. But upon performing IHC of the brain sections with the fluorophore at room
temperature, there was no staining observed. However, at 100 0C, the sections were stained
with this fluorophore. What could be the likely reason for this observation. (Note: There was
no change in the structural integrity of the fluorophore or its properties at these different
temperatures)
➢ Some possible reasons are-
1) It might be possible that during fixation, the binding sites of the protein for the
fluorophore got bound to the fixating agent or some other biomolecules present in
the tissue instead. At high temperatures these binding sites became vacant and
thus the fluorophore was able to bind. The binding sites might have become
vacant simply because of thermodynamic/kinetic reasons such that binding of the
fluorophore to X is more favored at high temperature. If the fixating agent was
PFA another possible reason can be that PFA decomposes into formaldehyde on
heating thus making the binding sites available for the fluorophore.
2) It is also possible that increased temperature made the cells of the tissue more
permeable to antibodies used in IHC.
3) Since, we are working with brain slices which have been fixed, it rules out the
possibility of the protein being synthesized only at high temperature as fixed
tissues are dead. Thus, there is the possibility of the presence of precursor protein
that undergoes some chemical change and protein X is produced at high
temperature. This change has to be purely thermodynamical and not based on
some enzymes as all enzymes have been inactivated by fixation.

Q3. A 27-year-old had male who had suffered severe seizures since childhood underwent
surgery in which a thumb-sized section of tissue from each side of his brain was removed in
an attempt to suppress the seizures. The seizures subsided, but the patient was left with
permanent amnesia. He could remember some things like scenes from his childhood, some
facts about his parents, and historical events that occurred before his surgery, but he was unable
to form new memories. He experienced every aspect of his daily life — eating a meal, taking
a walk — as a first. Yet his intellect, personality, and perception were intact, and he was able
to acquire new motor skills.

Answer the following questions based on the above description:

(i) On the basis of the symptoms and signs mentioned above, which tissue or part of the
brain do you think was removed?
(ii) According to you, how would this case have interested early neuroscientists and
contributed to the understanding of brain anatomy?
(iii) Discuss the major functions that are usually performed by the removed brain tissue
and mention any other clinical symptoms you think the patient could have posed
after its removal.
➢
i) Hippocampus of the medial temporal lobe was removed. Seizures generally
happen due to damage to the hippocampus. Hippocampus is involved in
forming new memories. Old memories are present in the cortex thus he
remembered old things but not things after the surgery. He could learn new
 motor skills (Procedural Memory) because Striatum is responsible for it. Cortex
is responsible for intellect, personality, and perception. Thus, they were intact.
ii) This case was interesting to early neuroscientists as he could be given different
tasks. His ability to not perform certain tasks would mean the hippocampus is
involved in those functions.
iii) Other major functions of Hippocampus-
1) It is involved in spatial learning and navigation. Thus, it is expected that
he would face difficulty in navigating.
2) It is involved in inhibitory control of responses. Thus, the patient should
also exhibit hyperactivity i.e., he will respond severely to even the
slightest of stimuli.
3) Hippocampus also possesses “Concept” cells which for e.g., help to relate
the image of Luke Skywalker to the image of the character. Thus, it is
expected that he will be unable to make such relations between words,
images etc. to a particular concept.

Q4. A patient with brain injury complains that he is experiencing numbness in certain parts of
his body and is unable to a identify objects by touch alone. During neurological examination,
he fails to distinguish whether an X or an O was written on the palm of his hand, however his
memory, intellect and personality didn’t show any significant difference from before the
accident. Adding to this, he also complaint that it is getting difficult for him to location the
positions of his body parts in space.

Answer the following questions based on the above description:

i. According to the mentioned neurological observations, which part of the brain has
suffered a lesion?
ii. Discuss the major functions that are usually performed by the lesioned brain area and
mention any other clinical symptoms (with brief reasoning) that the patient could have
shown because of the lesion.

➢
i) Clearly, his sense of touch has been affected. Also, proprioception (ability to
determine location of body parts) has been affected. Thus, the somatosensory
pathway is affected. Thus, the parietal lobe has suffered a lesion, particularly the
primary and/or secondary somatosensory cortex.
ii) Along with touch and body position, the somatosensory system is also involved in
the sensation of temperature, movement, pressure, and pain. Thus, he will not be
able to differentiate between hot and cold objects, but he will not get any burning
sensation as sense of pain has also been affected. He will also not be able to feel if
someone pinches him. Although he might not be able to feel a sharp/hot object, it is
expected that he will remove his body away from such object as reflex actions
involve only the spinal cord and not the brain. If the lesion is not strictly restricted
to this region and some adjacent regions like primary motor cortex (frontal lobe)
and auditory cortex (temporal lobe) also suffer lesion, it is expected that motor
functions and hearing functions will also get affected.