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NEUR 356 > Lecture 2: Neuroanatomical Methods > Flashcards

Lecture 2: Neuroanatomical Methods Flashcards

1
Q

Nissl stain developed by:

A

Franz Nissl (german neuropathologist)

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2
Q

Nissl stain allows researchers to (2):

A

(1): Distinguish between different cell types (such as neurons and glia),
(2): Study neuronal shapes and sizes

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3
Q

Nissl staining works by :

A

exploiting the chemical properties of certain dyes that
preferentially bind to NUCLEIC ACIDS, particularly RNA and DNA

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4
Q

Nissl stain uses :

A

BASIC ANILINE DYES such as CRESYL VIOLET, TOLUIDINE BLUE, or METHYLENE BLUE

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5
Q

The dyes used in Nissl staining are _ charged molecules that are attracted to _

A

The dyes used in Nissl staining are positively charged (basic) molecules that are attracted to negatively charged molecules

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6
Q

The primary targets of Nissl staining are:

A

The acidic components of neurons:

(1)RNA in the rough endoplasmic reticulum (Nissl bodies)

(2) DNA in the cell nuclei

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7
Q

The primary targets of Nissl Staining are the acidic components of neurons (2):

A

(1) RNA in the rough endoplasmic reticulum (Nissl bodies), which are rich in polyribosomes
(2) DNA in the cell nuclei

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8
Q

RNA in the rough endoplasmic reticulum are rich in:

A

polyribosomes

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9
Q

What are the two steps involved in the mechanism of staining?

A
  1. The basic dyes interact electrostatically (ionic interaction, not a covalent bond) with the
    negatively charged phosphate groups of RNA and DNA (ionic interaction, not a covalent bond).
  2. This results in the staining of Nissl bodies (aggregates of rough ER) within the cytoplasm and
    nuclei of neurons.
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10
Q

Nissl staining predominantly highlights

A

neurons over glial
cells (due to higher RNA concentration)

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11
Q

selective staining: Nissl staining predominantly highlights neurons over glial
cells because

A

neurons have a HIGHER ABUNDANCE OF ROUGH ER AND RIBOSOMES
due to their active protein synthesis

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12
Q

Why do we say that Nissl stains can selectively stain?

A

Nissl staining predominantly highlights neurons over glial
cells because neurons have a higher abundance of rough ER and ribosomes
due to their active protein synthesis

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13
Q

Visualization: Under a microscope, Nissl-stained sections show

A

neuronal cell
bodies with intensely stained Nissl substance and nuclei, while the surrounding
neuropil and non-neuronal structures remain less prominent

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14
Q

Neuropil:

A

the space between neuronal and glial cell bodies that
is comprised of dendrites, axons, synapses, glial cell processes,
and microvasculature.

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15
Q

Nissl stain application:

A

(1)studying NEURONAL ARCHITECTURE (2) IDENTIFYING CHANGES IN NEURONAL POPULATIONS in various regions of the nervous system.

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16
Q

“Nissl bodies” (aka (also called Nissl granules, Nissl substance or tigroid substance) are

A

portions of rough Endoplasmic
Reticulum studded with ribosomes

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17
Q

Golgi’s method is a

A

silver impregnation technique that is used to
visualize nervous tissue under light microscopy

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18
Q

Golgi’s method was discovered by:

A

Camillo Golgi - 1870s.

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19
Q

What are the name of the two steps involved in golgi staining?

A

1) fixation
2) impregnation

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20
Q

Describe the first step of golgi staining; FIXATION

A
  • The nervous tissue is initially fixed in a potassium dichromate solution to CREATE REACTIVE SITES
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21
Q

describe the second step of golgi staining: impregnation

A

** after fixation**

The tissue is then immersed in a solution of SILVER NITRATE (AgNO₃).

  • The SILVER NITRATE REACTS WITH THE DICHROMATE IONS
    to form
    INSOLUBLE SILVER CHROMATE (Ag₂CrO₄) PRECIPITATE (MICROCRYSTALLIZATION).
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22
Q

Golgi stain : selective staining: the silver chromate precipitate forms:

A

ONLY IN A SUBSET OF NEURONS and glial cells due to subtle variations in cellular properties –> ALLOWING DETAILED VISUALIZATION OF DENDRITES + AXONS

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23
Q

Golgi stain: this selective staining fills the __ allowing ___

A

This selective staining fills the CELL BODIES, DENDRITES and AXONS, allowing their morphology to be visualized in intricate detail

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24
Q

Visualization golgi stain : (COLOR + STABILITY?)

A

The silver chromate precipitate is dark brown to black and highly
insoluble, making it stable and suitable for microscopic analysis.

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25
Golgi staining was extensively used by:
Santiago Ramón y Cajal (1852–1934) (INSPIRED BIRTH OF NEURON DOCTRINE)
26
Golgi staining was extensively used by Spanish neuroanatomist Santiago Ramón y Cajal (1852–1934) to make __ , inspiring __
Golgi staining was extensively used by Spanish neuroanatomist Santiago Ramón y Cajal (1852–1934) to make fundamental discoveries about the organization of the nervous system, inspiring the birth of the NEURON DOCTRINE.
27
Can you visualize dendriti spines with golgi staining?
yes
28
one of the most powerful tools for studying the morphology of neurons and their connectivity
GOLGI STAINING
29
ability to label neurons in their entirety continues to provide critical insights into the structure of the nervous system.
Golgi Stain
30
Histochemical Techniques Definition:
Use biochemical reactions to visualize brain molecules.
31
What are two types of Histochemical techniques?
(1) Immunocytochemistry (2) In situ Hybridization
32
Immunocytochemistry (histochemical technique):
Antibodies highlight specific proteins, (e.g., membrane or nuclear proteins, neurotransmitters, etc)
33
In situ Hybridization (histochemical technique):
Detects active gene expression by binding RNA probes to mRNA.
34
Immunocytochemistry/Immunohistochemistry uses:
Antibodies to detect specific antigens (proteins, peptides, or other molecules) within cells or tissue sections.
35
Immunocytochemistry/ImmunohistoChemistry applications (3):
(1) Identifying specific cell types (e.g., neurons vs. glia) (2) Mapping distribution of neurotransmitters/enzymes/receptors (3) Studying pathological conditions (e.g., neurodegenerative diseases, tumors).
36
Histochemical Techniques: Immunocytochemistry/Immunohistochemistry: mechanism (2):
-Antibodies bind to target proteins (antigens) -Antibodies are conjugated with a detectable marker, such as fluorescent dues (FLUORESCENCE MICROSCOPY) or enzymes (horseradish peroxidase) → colored reaction under light microscopy
37
Histochemical Techniques Immunocytochemistry/Immunohistochemistry: Fluorescent dyes (e.g., FITC, Alexa Four) markers for :
Visualization under fluorescence microscopy
38
Histochemical Techniques Immunocytochemistry/Immunohistochemistry: Enzymes (e.g., horseradish peroxidase or alkaline phosphatase)markers produce:
a colored reaction product visible under light microscopy
39
40
Immunocytochemistry/Immunohistochemistry advantages (2) :
(1) High specificity and sensitivity. (2) Compatible with other techniques like confocal microscopy for 3D imaging.
41
Immunocytochemistry/Immunohistochemistry limitations (2) :
(1) Requires well-validated antibodies to ensure specificity. (2) Signal strength can vary depending on antigen abundance and accessibility.
42
In situ hybridization in neuroscience involves use of:
using a complementary mRNA strand to detect mRNA expression
43
In situ hybridization applications (2):
(1) Research: Gene expression (2) clinical: Brain tumour diagnosis, Schizophrenia research (identifying altered transcript localization)
44
In situ hybridization: what are the 5 steps:
FPHWD (1) FIXATION: preserves tissue structure and stabilizes nucleic acids. (2)PROBE DESIGNE: Probes are synthesized to target specific gene sequences and are labeled for detection. (3)HYBRIDIZATION: Probes are applied to the tissue and allowed to bind to complementary mRNA sequences. (4)WASHING: Removes unbound probes to reduce background signal. (5)DETECTION: Visualized using fluorescence microscopy, autoradiography, or other imaging techniques.
45
In situ hybridization How does it work?
– Synthetic RNA probes, labeled with a detectable marker (e.g., fluorescent or radioactive tags), bind (hybridize) to complementary target mRNA sequences. – The hybridization process allows visualization of gene expression patterns in their natural tissue context. – Can target mRNA or DNA, depending on the purpose
46
In situ hybridization:Can target mRNA or DNA, depending on the purpose: mRNA:
For detecting and visualizing active gene expression
47
In situ hybridization:Can target mRNA or DNA, depending on the purpose: DNA:
For detecting and localizing DNA sequences, such as specific genes or repetitive elements within the genome.
48
In situ hybridization Advantages (1):
High-resolution gene expression mapping
49
In situ hybridization:Limitations :
Time-intensive and requires careful probe design.
50
Brainbow Technique Uses:
genetically encoded fluorescent proteins to label neurons in multiple colors
51
Brainbow Technique enables:
detailed visualization of local connections and tiling between neurons using fluorescence microscopy.
52
Brainbow Technique: Applications (2):
(1) Single-cell resolution mapping of neural circuits. Each cell is labelled with a unique color, so that neurites can be individually traced (e.g. you can find out where a specific axon goes in order to understand how the circuit is connected). (2) Studying local connectivity
53
Single-cell resolution mapping of neural circuits
Brainbow Technique
54
Brainbow Technique: mechanism:
o DNA constructs with fluorescent proteins (e.g., GFP, CFP, YFP) are randomly recombined, producing unique neuron colors.
55
Optogenetics uses:
light-sensitive ion channels (e.g., channelrhodopsins) to control neuronal activity with light.
56
optogenic applications (2):
(1) Mapping neural connectivity (e.g., whether a set of neurons in area A excites or inhibits a downstream neuronal population in area B). (2) Understanding behavior-related neuronal activity
57
Viral-Based Tracing: Uses
modified viruses to trace synaptic connections
58
Herpes Simplex Virus (HSV) –
Multi-synaptic tracing.
59
Rabies Virus:
Retrograde transsynaptic tracing
60
Pseudorabies Virus (PRV) –
Traces from axon terminals to soma.
61
Dye-Based Tracing uses:
axonal transport to trace connections
62
Dye-Based Tracing: dyes used (3):
Horseradish Peroxidase (HRP) FluoroGold Dextrans
63
Dye-Based Tracing: Transport types:
Anterograde: Soma → Axon terminals. Retrograde: Axon terminals → Soma.
64
Magnetic Resonance Imaging (MRI):
STRUCTURAL and functional brain imaging.
65
Diffusion Tensor Imaging (DTI):
Visualizes AXONAL PATHWAYS via water diffusion.
66
Non-invasive Brain Imaging enable:
safe, IN-VIVO studies of human and animal brains.
67
Magnetic Resonance Imagine (MRI): Applications (2):
(1) Detecting and studying structural abnormalities (e.g., tumors, neurodegeneration). (2) Mapping cortical thickness and subcortical volume.
68
Magnetic Resonance Imagine (MRI) uses:
Uses hydrogen nuclei in water to generate high-resolution structural brain images
69
Magnetic Resonance Imaging (MRI):* Advantages (2)
o High spatial resolution (~1mm). o No ionizing radiation.
70
Magnetic Resonance Imaging (MRI):* Limitations:
o Expensive. o Claustrophobia & loud noise.
71
What is a Voxel
* A voxel (short for "volume pixel") is the smallest unit of 3D spatial resolution in MRI imaging. * Analogous to a pixel in 2D images, but represents a tiny cube of tissue in 3D space.
72
Magnetic Resonance Imagine (MRI) Voxel Size and Image Quality - Small Voxels provide:
higher spatial resolution.
73
Magnetic Resonance Imagine (MRI) Voxel Size and Image Quality - Small Voxels allow:
finer anatomical details to be visualized
74
Magnetic Resonance Imagine (MRI) Voxel Size and Image Quality - Small Voxels require:
longer scan times and higher signal-to-noise ratio (SNR).
75
Magnetic Resonance Imagine (MRI) Voxel Size and Image Quality - large Voxels
Reduce spatial resolution but improve SNR.
76
Magnetic Resonance Imagine (MRI) Voxel Size and Image Quality - large Voxels: scan time:
Faster scan times, suitable for broader overviews.
77
Brain Imaging: Magnetic Resonance Imagine (MRI) Advantages (2):
* Better spatial resolution (~1mm) compared with older methods (e.g., computerized tomography, CT’s resolution is 0.5 to 1 cm. * Does not use ionizing radiation.
78
Brain Imaging: Magnetic Resonance Imagine (MRI) limitations (4):
* Expensive * Metal implants and pacemakers contraindicated. * Claustrophobia * Loud noise
79
Brain Imaging: Diffusion Technique:
Specialized MRI that maps white matter tracts.
80
DTI mechanism:
Tracks anisotropic water diffusion along axons
81
Brain Imaging: Diffusion Tensor Imaging applications:
o Mapping brain connectivity. o Detecting axonal damage (e.g., stroke, multiple sclerosis).
82
Brain Imaging: Diffusion Tensor Imaging technique: direction axonal information flow?
The direction of axonal information flow cannot be distinguished with the method, and the resolution of the images does not extend to the cellular level.
83
Brain Imaging: Diffusion Tensor Imaging: particularly useful in:
picturing pathologies that affect axonal pathways (e.g., tumors that distort axon trajectories or strokes that destroy axon pathways).
84
Brain Imaging: Diffusion Tensor Imaging Limitations (3):
* Crossing Fibers: DTI struggles to resolve regions where multiple fiber bundles cross, as the ellipsoid model assumes one dominant direction per voxel. * Noise Sensitivity: Requires high signal-to- noise ratios for accurate measurements. * Limited Spatial Resolution: Cannot visualize individual axons; instead, it maps macroscopic bundles.
85
Computational Neuroanatomy Combines :
imaging techniques and computational tools for modeling and analysis
86
Quantitative Analysis:
Measuring brain volumes, cortical thickness, connectivity patterns, and other structural features
87
Machine Learning Algorithms:
Analyzing large datasets to identify patterns, classify brain regions, or predict disease progression.
88
Simulations:
Modeling neuronal activity and interactions to understand dynamic brain processes.
89
Computational Neuroanatomy Applications (3)
o Disease diagnosis (e.g., Alzheimer’s, Parkinson’s). o Studying brain plasticity and learning. o Machine learning for neural structure analysis.
90
Computational Neuroanatomy Applications: Research (2):
* Mapping structural and functional connectivity in the brain. * Understanding brain plasticity and its role in learning and memory.
91
Computational Neuroanatomy Advantages (3):
* Enables the integration of large-scale datasets for comprehensive analysis. * Provides insights into the relationship between brain structure and function. * Allows for predictive modeling of brain changes in health and disease.
92
Computational Neuroanatomy: challenges (3):
* Requires significant computational resources and expertise. * Data standardization and integration remain complex due to variability across studies. * Ethical considerations in handling sensitive patient data.

NEUR 356 (9 decks)

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