This seminar presentation summarizes research on chemoarchitectonics and neural growth factors. It discusses chemoarchitectonics as the study of brain anatomy and function through staining specific chemicals to identify neurotransmitter patterns. The presentation reviews a study on lungfish brains that identified neuronal populations and tracts using calbindin and calretinin staining. It also discusses Brodmann's cytoarchitectonic mapping of the cerebral cortex and Nissl staining technique. Finally, it provides an overview of neurotrophins like NGF, their discovery, biogenesis, functions in neuron survival and differentiation, and signaling mechanisms through neurotrophin receptors.

1. SEMINAR PRESENTATION ON:
CHEMOARCHITECHTONICS
AND
NEURAL GROWTH FACTORS
1

2. PRESENTERS
•OSHODI OLABODE
•NWOYE DAVID O.
•OBI-OJINIKA CHUKUBUEZE
•IKPA ONAH JAMES
2

3. INTRODUCTION
•Chemoarchitectonics is the study of the anatomy
and function of the brain and spinal cord through
the use of imaging, immunohistochemistry,
molecular & optogenetics, stem cell and cellular
biology, engineering, neurophysiology and
nanotechnology.
3

4. INTRODUCTION CONTD…
• Chemoarchitectonics deals with the use of
specific chemicals via staining to identify the
pattern of neurotransmitters or neuros in the NS
• It is broadly called cytoarchitectonics or brain
mapping
4

5. •Immunohistochemical studies on lungfishes have
proved that the detection of CALBINDIN (CB) and
CALRETININ (CR) provides a powerful method of
discerning segregated neuronal populations, fibre
tracts and neurophils (Morona et al., 2018)
5

6. •Chemoarchitecture has demonstrated to be a
useful tool for identifying subpopulations of
neurons generally not recognizable.
6

7. CYTOARCHITECTURE
•Korbinian Brodmann subdivided the cerebral
cortex into numerous areas based on regional
differences in the distribution, density, shape, and
size of cell bodies, i.e., the cytoarchitecture
(Brodmann, 1909).
•He was convinced that each cortical area
subserves a certain function within a larger
network.
•This led to Broadmans classification of the brain
into four main lobes
7

8. LOCALIZATION OF FUNCTION IN THE NERVOUS
SYSTEM: CYTOARCHITECTURE
K. Brodmann,1909
8

9. Investigators are using sophisticated technology to bring together cytoarchitectural and MRI-based
topographic mapping:
The Jülich–Düsseldorf atlas
Eickhoff, Amunts, Zilles
9

10. The neocortex (isocortex)
has six layers:
I. Molecular layer
II. External granular layer
III. External pyramidal layer
IV. Internal granular layer
V. Internal pyramidal layer
VI. Multiform layer
Subcortical white matter
III
II
I
IV
V
VI
Layer III Pyramidal neurons
Cresyl violet-stained section through the cerebral
cortex, showing its cytoarchitecture.
10

11. Summary of Cortical Layers & Their Connections
Corticostriates
11

12. • In present neuroimaging studies, Brodmann’s schematic
drawings of cortical maps are still frequently used
references to register functional activations to anatomical
structures, although his map does not match more recent
anatomical and functional data in many brain regions and
new approaches are mandatory (Zilles and Amunts, 2010)
12

13. •Other techniques like optical coherence
tomography visualizes the cortical laminar structure
to the individual neurons
13

14. •One of the commonly known staining methods in
brain chemoarchitecture is NISSL STAINING
14

15. THE NISSL STAINING TECHNIQUE
•The Nissl staining technique is commonly used for
determining the cytoarchitectonics of neuroanatomical
structures, using common agents such as thionin, cresyl
violet, or neutral red.
•These dyes intensely stain "Nissl bodies" (rough
endoplasmic reticulum), which are abundant in neurons
and reveal specific patterns of cytoarchitecture in the
brain.
15

16. •Other common staining techniques used by
histologists in other tissues (such as the hematoxylin
and eosin or "H&E stain") leave brain tissue appearing
largely homogeneous and do not reveal the level of
organization apparent in a Nissl stain.
16

17. •Nissl staining reveals details ranging from the
macroscopic, such as the laminar pattern of the
cerebral cortex or the interlocking nuclear patterns
of the diencephalon and brainstem, to the
microscopic
•such as the distinctions between individual neurons
and glia in any subregion of the central nervous
system.
17

18. •Many other neuroanatomic and cytoarchitectonic
techniques are available to supplement Nissl
cytoarchitectonics; Including immunohistochemistry and in
situ hybridization, which allow one to label any gene or protein
expressed in any group of cells in the brain.
18

19. •However, Nissl cytoarchitecture remains a reliable,
inexpensive, and familiar starting or reference point for
neuroscientists wishing to examine or communicate
their findings in a widely recognized anatomical
framework and in reference to neuroanatomical atlases
which use the same technique.
19

20. PAPER REVIEW
Regional chemoarchitecture of the brain of lungfishes based on
calbindin D-28K and calretinin immunohistochemistry
Ruth Morona, Jesús M. López, R. Glenn Northcutt and Agustín González
The Journal of Comparative Neurology
Research in Systems Neuroscience
DOI 10.1002/cne.24422
© 2018 Wiley Periodicals, Inc.
20

21. INTRODUCTION
• Calbindin-D28k (CB) and calretinin (CR) are members of the
EF-hand calciumbinding proteins that are expressed widely
in neuronal cell bodies, axons and synaptic terminals
throughout the vertebrate nervous system.
• They function as fast calcium buffers to regulate cell
excitability and release of synaptic vesicles
• The physiological functions of these protein are not fully
known are not fully known
21

22. METHODOLOGY
• Seven (7) juvenile African lungfish protopterus dolloi
(total length 21-27cm) and five (5) juvenile Australian
lungfish, Neoceratodus fosteri (total length 35-42cm)
were used
• Sexes for the Australian lungfish were not established
but 4 males and 3 females of the African lung fishes
were established
• All animals were maintained in aquaria at 24-28º C
under natural light conditions.
22

23. • The animals were deeply anesthetized by immersion in 0.01% tricaine
methanesulfonate solution and perfused transcardially with
physiological saline followed by 200 ml of cold 4% paraformaldehyde in
a 0.1 M phosphate buffer (PB, pH 7.4).
• The brains were removed and kept in the same fixative for 2- 3 hours.
• Subsequently, they were immersed in a solution of 30% sucrose in PB
for 4-6 hours at 4 °C until they sank, then embedded in a solution of
20% gelatin with 30% sucrose in PB, and stored for 6 hours in a 3.7%
formaldehyde solution at 4 °C.
23

24. •The brains were cut on a freezing microtome at 30-
40μm in the transverse or sagittal plane, and
sections were collected and rinsed in cold PB.
• Bright field immunohistochemistry and double
immunohistoflourescence were carried out
24

25. RESULTS
•The antibodies against CR and CB used in the
present study revealed patterns of immunostaining
that, for each of the two species examined, were
constant from animal to animal, and no indications of
sex differences were observed.
•Comparison of identically processed brain sections
from Neoceratodus forsteri and Protopterus dolloi
showed mostly similar results
25

26. • The general characteristics of the observed labeling in the
main brain subdivisions, from rostral to caudal. The study of
sagittal and “classical” transverse sections helped to clarify the
localization and morphology of positive neurons and the
distribution of their dendritic processes.
26

27. •The CB/CR doubly labeled sections were used to
highlight the degree of their colocalization, and the
combinations with TH and ChAT mainly served to
evaluate the actual position of certain cell groups in
the brain within the segmental model
27

28. POINTS TO NOTE
• The study is basically for brain mapping
•The present study is the first that provides a
comprehensive and detailed map of the distribution of
the cells and fiber systems that contain CB and CR in
the brain of lungfishes.
•Comparison with other vertebrates reveals that
lungfishes share most of their features of calcium
binding protein distribution with amphibians,
particularly with salamanders.
28

29. • In turn, the results in lungfishes compared to those in
other fish groups, mainly actinopterygians, show
marked differences.
• The present data, in general, show that major traits in
the organization of the CB and CR neuronal systems are
shared among sarcopterygians and provide substantial
anatomical evidence to support the idea that living
lungfishes are close relatives to tetrapods
29

30. • Austrailian lungfishes sexes were not specified in the work
• The Australian lungfishes were perfused and fixed in carlifornia then transported to
spain
30

31. NEUROTROPHINS
•The neurotrophins are a family of proteins that are
essential for the development of the vertebrate
nervous system.
31

32. • Members of this family include;
 NGF – Nerve growth factor
 BDNF – Brain-derived neurotrophic factor
 NT3 – Neurotrophin 3
 NT4 – Neurotrophin 4
32

33. HISTORICAL PERSPECTIVE
• NGF was discovered through a series of experiments in
the 1950s on the development of the chick nervous
system.
• Since its discovery, NGF has been found to act in a variety
of tissues throughout development and adulthood.
33

34. • Rita Levi Montalcini and Viktor Harmburger.
• Stanley Cohen a Biochemist successfully purified NGF.
• He also determined the factor to be mostly composed of
protein with some nucleic acids.
34

35. •The discovery of NGF led to an award of the Nobel
Prize in Physiology or Medicine to Rita Levi-
Montalcini and Stanley Cohen in 1986.
35

36. BIOGENESIS
• The neurotrophins are initially synthesized as precursors or
pro-neurotrophins, which are cleaved to produce the
mature proteins. (ProNGF -> NGF)
• Pro-neurotrophins are cleaved intracellularly by FURIN or
pro-convertases at a highly conserved dibasic amino-acid
cleavage site to release carboxy-terminal mature proteins.
36

37. • The mature proteins:
• about 12 kDa in size, form stable, non-covalent dimers,
• normally expressed at very low levels during development.
• The amino-terminal half (or pro-domain) of the pro-
neurotrophin is believed to be important for the proper
folding and intracellular sorting of neurotrophins.
37

38. FUNCTIONS
• Signaling particular cells to survive, differentiate, or grow.
• Preventing the associated neuron from initiating
programmed cell death(PCD) - thus allowing the neurons to
survive.
• Neurotrophins also induce differentiation of progenitor cells,
to form neurons.
38

39. • It has been implicated in immune function,
stress response, nerve maintenance, and in
neurodegenerative diseases.
39

40. MECHANISM OF ACTION. NEUROTROPHIN RECEPTORS
•Each neurotrophin can signal through two different types
of cell surface receptors:
•Trk receptor tyrosine kinases
•p75 neurotrophin receptor (p75NTR).
• Transmembrane receptor, also known as TNFRSF16.
• a member of the tumour necrosis factor receptor (TNFR)
death-receptor family.
40

41. MODELS OF TRK AND P75 RECEPTOR
•Different neurotrophins show
binding specificity for particular
receptors.
•These interactions have
generally been considered to be
of high affinity.
Chao MV. 2004
41

42. • 1
42

43. Chao MV. 2004
43

44. CONCLUSION
• Neurotrophins remain important in shaping the structure of
connections even in mature brain especially in considering
to how the brain respond to injuries and diseases.
• Further studies on neurotrophins may pave way for the
pathological basis of many neurological diseases
44

45. 45

46. AIM OF STUDY:
•To determine the effect of BNN-27 on
photoreceptive survival after experimental
retinal detachment (RD).
46

47. EXPERIMENT/METHOD:
•BNN27 was administered intraperitoneally.
• Animals received one injection of BNN27
(200 mg/kg, diluted in 6% absolute ethanol in
water) or vehicle (6% absolute ethanol in
water) one hour post RD
47

48. •A total of seven injections of BNN27 (200mg/kg,
diluted in 6% absolute ethanol in water) or
vehicle (6% absolute ethanol in water) starting one
hour post RD and then administered once daily.
48

49. EFFECTS OF BNN-27 (STRENGTH):
• TUNEL+ photoreceptors were signifcantly decreased
24hours post injury after a single administration of
200mg/kg BNN27.
• Furthermore, BNN27 increased infammatory cell
infltration, as well as, two markers of gliosis 24hours
post RD.
49

50. •However, single or multiple doses of BNN27 were not
able to protect the overall survival of photoreceptors
7 days post injury.
•Additionally, BNN27 did not induce the
activation/phosphorylation of TrkAY490 in the
detached retina although the mRNA levels of the
receptor were increased in the photoreceptors post
injury.
50

51. WEAKNESS:
• One of the major disadvantages in this research is that,
Together these findings, do not demonstrate
neuroprotective activity of BNN27 in experimentally induced
RD.
• Their report states that C57BL/6 male mice (7–10 weeks)
were purchased for this experiment but we don't know the
population size.
51

52. • From their report the were kept in a 12hr light and 12hr
dark cycle, but they fail to tell us their observation
within the 7days duration they carried out the
experiment.Weight of animals were not reported, it
could be that the animals were over or under weight
for the dosage administered.
52

53. Thank you.
53

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