Lecture 2 Flashcards
Nissl stain developed by:
Franz Nissl (german neuropathologist)
Nissl stain allows researchers to:
distinguish between different cell types (such as neurons and glia), and neuronal shapes and sizes, in various regions of the nervous system
Nissl Stain requires:
preprocessing of the brain tissue
Nissl staining works by :
exploiting the chemical properties of certain dyes that
preferentially bind to NUCLEIC ACIDS, particularly RNA and DNA
Nissl stain uses :
BASIC ANILINE DYES such as CRESYL VIOLET, TOLUIDINE BLUE, or METHYLENE BLUE
The dyes used in Nissl staining are _ charged molecules that are attracted to _
The dyes used in Nissl staining are positively charged (basic) molecules that are attracted to negatively charged molecules
The primary targets of Nissl staining are:
The acidic components of neurons:
(1)RNA in the rough endoplasmic reticulum (Nissl bodies)
(2) DNA in the cell nuclei
The primary targets of Nissl Staining are the acidic components of neurons (2):
(1) RNA in the rough endoplasmic reticulum (Nissl bodies), which are rich in polyribosomes
(2) DNA in the cell nuclei
RNA in the rough endoplasmic reticulum are rich in:
polyribosomes
What are the two steps involved in the mechanism of staining?
- 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). - This results in the staining of Nissl bodies (aggregates of rough ER) within the cytoplasm and
nuclei of neurons.
Nissl staining predominantly highlights
neurons over glial
cells
selective staining: 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
Why do we say that Nissl stains can selectively stain?
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
Visualization: Under a microscope, Nissl-stained sections show
neuronal cell
bodies with intensely stained Nissl substance and nuclei, while the surrounding
neuropil and non-neuronal structures remain less prominent
Neuropil:
the space between neuronal and glial cell bodies that
is comprised of dendrites, axons, synapses, glial cell processes,
and microvasculature.
Nissl stain application:
studying NEURONAL ARCHITECTURE and IDENTIFYING CHANGES IN NEURONAL POPULATIONS in various regions of the nervous system.
Nissl bodies can also be called (3):
Nissl granules
Nissl substance
tigroid substance
“Nissl bodies” (aka (also called Nissl granules, Nissl substance or tigroid substance) are
portions of rough Endoplasmic
Reticulum studded with ribosomes
Golgi’s method is a
silver impregnation technique that is used to
visualize nervous tissue under light microscopy
Golgi’s method was discovered by:
Camillo Golgi, an Italian physician and scientist,
in the 1870s.
What are the name of the two steps involved in golgi staining?
1) fixation
2) imregnation
Describe the first step of golgi staining; FIXATION
- The nervous tissue is initially fixed in a potassium dichromate solution.
- Potassium dichromate (K₂Cr₂O₇) dissociates into potassium (K⁺),
DICHROMATE IONS (Cr₂O₇²⁻), and hydrogen ions in water. - The dichromate ions interact with cellular components, partially oxidizing
them and CREATING REACTIVE SITES, particularly in the membranes of some
neurons
describe the second step of golgi staining: impregnation
** after fixation**
The tissue is then immersed in a solution of SILVER NITRATE (AgNO₃).
- The SILVER NITRATE REACTS WITH THE DICHROMATE IONS and/or with the reduced
cellular components (produced during the oxidation step) to form
INSOLUBLE SILVER CHROMATE (Ag₂CrO₄) PRECIPITATE (MICROCRYSTALLIZATION).
Golgi stain : selective staining: the silver chromate precipitate forms:
ONLY IN A SUBSET OF NEURONS and glial cells due to subtle variations in cellular properties
Golgi Stain: Selective Staining:The silver chromate precipitate forms only in a subset of neurons
and glial cells due to:
subtle variations in cellular properties, such as
membrane composition and oxidation states, that affect the
chemical reaction.
Golgi stain: this selective staining fills the __ allowing ___
This selective staining fills the CELL BODIES, DENDRITES and AXONS, allowing their morphology to be visualized in intricate detail
Visualization golgi stain:
The silver chromate precipitate is dark brown to black and highly
insoluble, making it stable and suitable for microscopic analysis.
Visualization golgi stain: surrounding:
Surrounding unstained tissue remains largely transparent, creating
excellent contrast and enabling detailed visualization of neural
structures.
Golgi staining was extensively used by:
Spanish neuroanatomist
Santiago Ramón y Cajal (1852–1934)
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.
Can you visualize dendriti spines with golgi staining?
yes
Golgi staining remains
one of the most powerful tools for studying
the morphology of neurons and their connectivity
ability to label neurons in their entirety continues to provide
critical insights into the structure of the nervous system.
Golgi Stain
Staining in neocortex: golgi staining shows
Cell bodies & processes
Staining in neocortex: Nissl staining shows:
mostly cell bodies
Staining in neocortex: myelin staining shows:
myelinated axon
Histochemical Techniques Definition:
Use biochemical reactions to visualize brain
molecules.
What are two types of Histochemical techniques?
(1) Immunocytochemistry
(2) In situ Hybridization
Immunocytochemistry (histochemical technique):
Antibodies highlight specific proteins,
(e.g., membrane or nuclear proteins, neurotransmitters, etc)
In situ Hybridization (histochemical technique):
Detects active gene expression by
binding RNA probes to mRNA.
Immunocytochemistry/Immunohistochemistry uses:
Antibodies to detect specific antigens (proteins, peptides, or other molecules) within cells or tissue sections.
Immunocytochemistry/Immunohistochemistry applications (4):
(1) Identifying specific cell types (e.g., neurons vs. glia) by targeting cell-specific proteins.
(2) Mapping the distribution of neurotransmitters, enzymes,
or receptors in the nervous system.
(3) Studying cellular responses in normal and pathological
conditions (e.g., neurodegenerative diseases, tumors).
(4) Co-localizing multiple proteins in the same cell or tissue
section using different fluorophores.
Histochemical Techniques: Immunocytochemistry/Immunohistochemistry: mechanism:
Antibodies are designed to bind specifically to a
target molecule (antigen) in the tissue.
Antibodies are conjugated with a detectable marker, such as fluorescent dues , enzymes
Fluorescent dyes (e.g., FITC, Alexa Four) for :
Visualization under fluorescence microscopy
Enzymes (e.g., horseradish peroxidase or
alkaline phosphatase) produce:
a colored
reaction product visible under light microscopy
Immunocytochemistry/Immunohistochemistry advantages (3) :
(1) High specificity and sensitivity.
(2) Allows localization of molecules at a subcellular level.
(3) Compatible with other techniques like confocal
microscopy for 3D imaging.
Immunocytochemistry/Immunohistochemistry limitations (2) :
(1) Requires well-validated antibodies to ensure specificity.
(2) Signal strength can vary depending on antigen
abundance and accessibility.
In situ hybridization in neuroscience involves:
using a complementary mRNA
strand to detect and visualize the spatial distribution of
specific mRNA sequences within tissue samples, providing
insights into gene expression patterns in the nervous system.
In situ hybridization applications (3):
(1) Gene expression: to identify which neurons express a gene
(and therefore a target peptide or protein)
(2) Brain tumor diagnosis: to diagnose brain tumors in formalin-
fixed paraffin-embedded (FFPE) samples
(3) Schizophrenia research: to identify the localization of
transcripts associated with schizophrenia risk in the human
brain
In situ hybridization: what are the 5 steps:
– Tissue preparation: Fixation preserves tissue structure
and stabilizes nucleic acids.
– Probe design: Probes are synthesized to target specific
gene sequences and are labeled for detection.
– Hybridization: Probes are applied to the tissue and
allowed to bind to complementary mRNA sequences.
– Washing: Removes unbound probes to reduce
background signal.
– Detection: Visualized using fluorescence microscopy,
autoradiography, or other imaging techniques.
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
In situ hybridization:Can target mRNA or DNA, depending on the purpose: mRNA:
For detecting and visualizing active gene expression
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.
In situ hybridization
Advantages (2):
(1) ISH provides a high-resolution snapshot of the
distribution of target transcripts
(2) ISH can be used on fixed and unfixed tissues.
In situ hybridization:Limitations (3):
(1)Requires well-validated antibodies to ensure specificity.
(2) Signal strength can vary depending on antigen abundance and accessibility.
(3)Time-intensive and requires careful probe design.
Brainbow Technique Uses:
the expression of variable amounts of
red, green, and blue fluorescent proteins in
neurons.
Brainbow Technique creates:
a combinatorial palette of colors,
allowing each neuron to be uniquely tagged.
Brainbow Technique enables:
detailed visualization of local
connections and tiling between neurons using
fluorescence microscopy.
Brainbow Technique: Applications (2):
– Mapping neural circuits with single-cell
resolution.
– Studying structural organization in complex
neural networks.
- Magnetic Resonance Imaging
(MRI):
Structural and functional
brain imaging.
Diffusion Tensor Imaging (DTI):
Visualizes axonal pathways via
water diffusion.
Non-invasive Brain Imaging enable:
safe, in-vivo studies of
human and animal brains.
Magnetic Resonance Imagine (MRI) provides:
high-resolution images of brain
structure, allowing for the study of brain
regions and their boundaries, cortical
thickness and surface area, volume of
subcortical structures
Magnetic Resonance Imagine (MRI): Applications (3):
- Identifying structural abnormalities in
neurological conditions (e.g., tumors, brain
injuries, neurodegenerative diseases). - Monitoring changes in brain morphology
during development, aging, or following
interventions (e.g., therapy, medication). - Supporting neurosurgical planning by mapping
brain anatomy precisely.
Magnetic Resonance Imagine (MRI)
utilizes:
the magnetic properties of hydrogen
nuclei in water molecules to generate detailed
images of brain anatomy.
Brain Imaging: Magnetic Resonance Imagine (MRI): detailed steps (3)
– A strong static magnetic field aligns the spins of hydrogen nuclei.
– Radiofrequency (RF) pulses temporarily disturb this alignment
The H nuclei emit RF signals as they realign with the
magnetic field.
– The emitted signals vary depending on the tissue type (gray matter, white matter, or cerebrospinal fluid), enabling
differentiation.
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.
Magnetic Resonance Imagine (MRI)
Voxel Size and Image Quality - Small Voxels provide:
higher spatial resolution.
Magnetic Resonance Imagine (MRI)
Voxel Size and Image Quality - Small Voxels allow:
finer anatomical details to be
visualized
Magnetic Resonance Imagine (MRI)
Voxel Size and Image Quality - Small Voxels require:
longer scan times and higher
signal-to-noise ratio (SNR).
Magnetic Resonance Imagine (MRI)
Voxel Size and Image Quality - large Voxels
Reduce spatial resolution but improve
SNR.
Magnetic Resonance Imagine (MRI)
Voxel Size and Image Quality - large Voxels: scan time:
Faster scan times, suitable for broader
overviews.
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.
Brain Imaging: Magnetic Resonance Imagine (MRI)
limitations (4):
- Expensive
- Metal implants and pacemakers contraindicated.
- Claustrophobia
- Loud noise
Brain Imaging: Diffusion Tensor Imaging technique used to:
image the white matter
of the brain.
Brain Imaging: Diffusion Tensor Imaging allows:
reconstruction of major axonal
pathways (fiber tractography).
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.
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).
Brain Imaging: Diffusion Tensor Imaging
Applications (3):
- Brain Connectivity: Mapping structural
connectivity by identifying major white
matter pathways. - Clinical Diagnostics:
– Detecting microstructural damage in
diseases like multiple sclerosis,
Alzheimer’s disease, and stroke.
– Monitoring developmental changes in
children and degeneration in aging. - Neuroscience Research: Understanding
brain plasticity and network organization
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.
Computational Neuroanatomy Combines :
advanced imaging techniques with
computational tools to model, quantify, and analyze
the structure and function of the nervous system
Computational Neuroanatomy: Key techniques (4):
- 3D Reconstructions
- Quantitative Analysis
- Machine Learning Algorithms
- Simulations
3D Reconstructions:
Creating detailed models of brain
regions or entire neural networks using imaging data
(e.g., MRI, CT scans, or electron microscopy).
Quantitative Analysis:
Measuring brain volumes,
cortical thickness, connectivity patterns, and other
structural features
Machine Learning Algorithms:
Analyzing large datasets
to identify patterns, classify brain regions, or predict
disease progression.
Simulations:
Modeling neuronal activity and
interactions to understand dynamic brain processes.
Computational Neuroanatomy
Applications
Clinical (2):
- Diagnosing and monitoring neurological diseases like
Alzheimer’s, Parkinson’s, and epilepsy. - Studying structural and functional changes in the brain
during aging, development, or recovery from injury.
Computational Neuroanatomy
Applications: Research (@):
- Mapping structural and functional connectivity in the
brain. - Understanding brain plasticity and its role in learning
and memory.
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.
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.
