Key takeaways U1 Flashcards
Types of cells in the human brain
Neurons, glia.
Cells lining the blood vessels, cells that make up
the meninges, cells that make cerebrospinal fluid,
pituitary cells that secrete hormones into the blood
The human brain contains approx how many neurons
86 billion neurons.
Xenopus brain contains how many neurons
Approx 16 million neurons
Mammals with big forebrains are…
gyrencephalis (have gyro and sulci)
Mammals with small forebrains are…
Lissencephalic (smooth surfaced forebrain)
Xenopus
veterbrate “model organism”
Benefit of vertebrate model organisms (or model organisms more broadly)
allow us to
study features of the nervous system that are evolutionarily conserved (basic features like
the origin of neurons from the neural plate/tube), but easier to study for a variety of
reasons: e.g. external development (instead of inside the mother) like Xenopus, availability of genetic and viral tools like mice, highly developed primate visual system like Rhesus
monkeys.
Abilities of model organisms that we do not have
locating moving prey
using echolocation (bats), not feeling pain (naked mole rats), detecting electric fields (electric fish),regenerating the adult nervous system (newts) or experimental advantages like connectomes (worms and flies)
Organisation of the vertebrate brain
Sensory information enters the brain via the cranial
nerves (olfactory, optic, trigeminal (mixed), facial, auditory vestibular and vagus. The other
cranial nerves are purely motor (oculomotor, trochlear, abducents, hypoglossal, spinal
accessory) or mixed (trigeminal, facial, glossopharyngeal, vagus,)
Sensory cranial nerves
olfactory, optic, vestibulocochlear (auditory)
mixed cranial nerves
Trigeminal, facial, glossopharyngeal, vagus
Motor cranial nerves
oculomotor, trochlear, abducens, spinal accessory, hypoglossal
Neuraxis in Xenopus vs primate brain
In Xenopus, linear. In primate brain, it is curved anterior to the hindbrain so that a cross section through the human forebrain is called coronal (parallel to the face).
List 3 types of neurons in the CNS
Sensory: cell body outside of the CNS, axon enters
the CNS to synapse. Motor: cell body inside of the
CNS. Axon exits the CNS and synapses on muscle (or
autonomic ganglia, collections of cell bodies that
innervate the viscera). 3) Interneuron.
Symmetrical sections
Coronal/transverse or horizontal
Asymmetrical sections
Saggital
Location of ventricles
Lateral ventricles (I and II) are anterior
VI ventricle is posterior
III ventricle is ventral.
Horizontal sections
Taken parallel to the rostral caudal axis of the brain. Parallel to the ground. (if standing upright)
Sagittal sections
Sections taken in the plane diving the two hemispheres.
Coronal/frontal sections
Sections in the plane of the face.
Transverse section
Perpendicular to the long axis of the brainstem and spinal cord. Dorsal ventral and posterior/anterior axis are the same
Longitudinal section
Parallel to the long axis of the brainstem and spinal cord.
Axes of the brain and spinal cord
Anatomical location of parts of the CNS is established when and how
Established during embryonic development. Molecular signals in the embryo convey positional info. TF expression in embryonic cells enables response to these signals.
How does the neural tube form in the embryo
The neural tube forms dorsally in the embryo. The lumen in the middle (medial) will form the
ventricles.
The neural tube transforms into the brain (anterior) and the spinal cord (posterior) during
development.
Signaling molecules provide positional information (anterior/posterior, dorsal/ventral for example). [medial/lateral usually due to cell migration.
How are signalling molecules decoded
Signaling molecules are decoded by transcription factors expressed in cell receiving the signal. Transcription factors are DNA binding molecules
Hox genes
TFs. The hox genes themselves and their positional code are highly conserved in both
invertebrates (the fruit fly) and vertebrates (humans). The order of hox genes on the
chromosome = the order (anterior to posterior aka rostral-caudal) in the neural tube region that becomes the
spinal cord.
Role of signalling molecules and TF expression on spinal cord, hindbrain and forebrain
Signaling molecules and the pattern of TF expression also pattern the developing spinal cord, the hindbrain and the forebrain.
Dorsal/ventral patterning:
Sonic hedgehog (Shh) released from the notochord induces the floorplate to itself produce and release Shh.
Bone morphogenetic protein (BMP)
released from the roofplate similarly “dorsalizes” the developing neural tube and the brain (D)
Relevance of the bauplan
Because the basic “bauplan” is conserved across vertebrates, we can study early formation of
the CNS in animals with external fertilization like fish and frogs.
Advantage of the Xenopus oocyte
The Xenopus oocyte (unfertilized egg, a sigle cell) is handy because it is color coded (black:
animal pole; white (vegetal pole).
Cells in the early embryo (fruit flies or vertebrates) have two sources of information:
1) intrinsic. (e.g. mRNAs localized in the oocyte that end up in different cells after cell division
starts (cell division is triggered by fertilization: the sperm entering the oocyte).
One example
is the mRNA for vg1 (vegetal 1) that helps to localize germ (sperm or egg) cell origins to the
ventral pole using microtubules as “railroad tracks”. Some mRNAs are localized to the animal pole. These include mRNAs
that code for factors preventing premature translation of mRNAs into protein i.e.
proteosome). These mRNAs are localized using the endoplasmic reticulum (membranous
tubules continuous with the nuclear membrane) that facilitate mRNA translation into protein.
2) Localization of mRNA helps to “fate map” the oocyte and determine which regions will
contribute to different parts of the body.
Extrinsic info that affects developing embryo
Cells in the developing embryo also have access to “extrinsic” information from nearby cells. This information changes over time because of movement.
a) When the sperm enters the egg, the outer “rind” of cytoplasm rotates relative to
internal oocyte cytoplasm i.e. intracellular components move “globally” (picture the Earth’s continents moving relative to its internal core, if that helps). This cortical rotation moves the position of maternally localised mRNAs.
b) During later blastula (many cells) stages, cells on the outside of the embryo begin to
move inside both dorsally and ventrally through an opening called the “blastopore”. They stream inside obliterating an internal cavity (the blastocoel) and creating a new cavity (the archenteron).
Gastrulation - what does it create
Gastrulation creates both anterior posterior and ventral/dorsal signaling to pattern the
embryo. Migrating cells form a layered structure that creates the “neurectoderm”, the part of
the embryo that will form the CNS (vertical signaling). Through lateral (planar) signaling from
the dorsal lip of the blastopore, the anterior-posterior axis of the SNS is established.
Formation of neural tube
Cues from location signal neural identity.
The neural groove forms on the dorsal side of the embryo, its sides start to round up and then
fuse to form the neural tube (covered by ectoderm). At this stage there are blocks of tissue
(the somites that rise to muscle and bone) on either side (lateral) of the neural tube. Some
cells get left out of the neural tube when it closes; these also give rise to neurons (and other
cell types) and are called the “neural crest”.
Placodes
The other source of nerve cells are “placodes”, thickened regions on the outside of the
embryo that can give rise to many cell types (lens of the eye) including sensory ganglia
(collections of nerve cell bodies outside of the CNS).
Role of signalling molecule gradients in spinal cord
Gradients of signaling molecules establish the identity of neurons in the spinal cord.
Interneurons that convey sensory information within the spinal cord are dorsal; motor
neurons that synapse on muscle fibers are ventral; other interneurons in the spinal cord form local neural circuits that create and coordinate movements (and posture).
Primary sensory neurons location
The cell bodies of primary sensory neurons are OUTSIDE of the CNS (usually in
groups that form “ganglia”); they send processes through the dorsal “roots” into the CNS and
contact sensory interneurons. The cell bodies of primary motor neurons are INSIDE of the
CNS (in the ventral “horn”). They send axons out through “ventral” roots to synapse on
muscle fibers (see Lecture 1).
Patterning of the hindbrain
Hox genes also allow signaling molecules to pattern the hindbrain including the identity of
the cranial nerves.
How is the developing neural tube transformed into the CNS (brain and spinal cord)?
Cell
proliferation and cell migration.
Cell proliferation
Cells begin to divide at the neural tube stage. Each cell is attached on one side to the lining of the “lumen” (central canal) and on the other side to the outside of the neural tube covered by a membrane (the pia or pa mater). The nucleus of the cell bobs back and forth. DNA
replicates as the nucleus moves towards the pial surface and the nucleus divides as it moves
towards the ventricular surface. There the cell detaches from the pila surface and partitions
the nuclei into two “daughter” cells each of which can go back into the cell cycle or migrate
away as a neuron. The exact moment the cell becomes a neuron (and will not divide again) is
called the “birthday”.
Neuron migration
Different brain regions have different migration patterns. In spinal cord (not shown) the
earliest “born” neurons migrate laterally so the oldest neurons are lateral and the youngest
ones are medial. In cortex, some neuroblasts migrate to the pial surface and make new
neurons that migrate inwards (neurons in cortical layer one). Most newly borned neurons
migrate out radially with the oldest neurons stopping close to the lumen and the younger
ones migrating past the (the “inside out pattern). A pulse of radioactive thymidine is taken
up by dividing cells but only accumulates (and can be detected at later times) on the cell’s
birthdate (see above).
What do newly “born” neurons do
Newly “born” neurons migrate on the processes of radial glial cells. The processes span the
luminal and pial surfaces. Radial glial cells themselves can detach and become neuroblasts.
Importance of migration
Migration is very important for normal function and many neurological disorders are
suspected to result from defects in this process.
Neural crest
Neural crest cells can give rise to many cell types and not just neurons; for example, sensory
neurons, cells in the adrenal gland, pigment cells (melanocytes). They also give rise to
structural components that determine the shape of the face. In humans, deficits in neural
crest cell development give rise to disorders of the palate and lips. When breeders select for
“pushed in” faces in dogs (such as pugs), they may sometimes also select for abnormal
development of the face and lips that hinder nursing.
Some placodes in the developing mouse embryo.
The eye ball’s origin
The “ball” originates from the otic placode. The retina is an outpouching of the forebrain
(diencephalon). The lens (otic placode) is induced to form by interaction between optic
vesicle (diencephalon) and ectoderm.
Migration of neurons in the developing cerebral cortex
In the developing cerebral cortex neurons migrate from the ventricular to the pial surface
using the processes of radial glial cells.
Neurites
The young neuron extends neurites, one at each pole of the cell. The neurites end in a growth cone, a specialized part of the neurite that samples the environment.
The tips of the neurites express express Par, a polarity scaffolding protein. One
neurite becomes an axon that expresses tau (a cytoskeletal protein; panel lower left) in the
neurite and Par at its tip. If Par is knocked out early, no neurites form (green ball). If Par is
knocked our later, all of the neurites become axons.
Growth cones
Growth cones move through the environment, sampling attractive and repulsive cues.
Growth cones follow attractive cues but collapse when they encounter repulsive cues (e.g.
semaphorin). Both responses are mediated by calcium levels in the growth cone.
Cells near the pill surface…
Cells near the pial surface convert semaphorin into an attractive molecule and the
developing apical dendrite is attracted. Semaphorin (and Slit) repel the axon. Exposure to localized signaling molecules - Slit, BDNF and Notch - promote growth of basal dendrites.
What promotes growth of basal dendrites
Exposure to localized signaling molecules - Slit, BDNF and Notch
Cresyl violet
a Nissl stain that
reflects cytoplasmic RNA.
Commissural neurons of the spinal cord
In the neural tube, the region that will become the spinal cord includes commissural neurons,
neurons whose axons travel ventrally and cross to the opposite (contralateral) side just above
the floor plate. These growth cones are attracted to the ventral midline by netrin and cross to
the opposite side. During crossing they lose the ability to respond to netrin so they don’t cross
back.
Commissural neurons of the spinal cord
In the neural tube, the region that will become the spinal cord includes commissural neurons,
neurons whose axons travel ventrally and cross to the opposite (contralateral) side just above
the floor plate. These growth cones are attracted to the ventral midline by netrin and cross to
the opposite side. During crossing they lose the ability to respond to entrain so they don’t cross back
If netrin is knocked out in a mouse embryo
If netrin is knocked out in a mouse embryo, interneuron axons do not cross the ventral
midline or fasiculate (bundle) together.
Motor neuron axons in the developing spinal cord
in the developing spinal cord of an early Xenopus tadpole, primary motor
neurons (the big cell) send axons out of the spinal cord into the somites. Secondary motor
neuron axons born later (lower panel) fasiculate with the pioneer axon and innervate muscles
of the trunk that allow the tadpole to hatch and swim.
Ontogenetic cell death
More motor neurons are generated during development than survive. Those that do not
connect to muscle targets undergo ontogenetic cell death (illustrated in a chick embryo). If the
limb bud is removed there is more cell death; if an extra limb bud is grafted, there is more cell
survival due to neurorophic factors (TFs). Ontogenetic cell death also accounts for sex
differences in the motor neurons that innervate specialized muscle supporting male
reproduction in rodents (SNB). No sex difference between males and females in other
motor neuron groups (RDLN, DLN).
Motor, sensory, vs interneurons
Sensory neurons (3): Cell bodies outside of CNS. Send axon into spinal cord
Motor neurons: (1) Cell bodies inside of CNS. Send axon into muscles
Interneurons(3): Cell bodies and processes (dendrites and axons) inside of CNS.
Neurons have molecular identities
Why is the mouse the most widely used model organisms in neuroscience
for many
reasons including its phylogeny (rodents and mammals as are humans) and the resources
available for experimentation, different mouse lines and the availability of detailed brain
atlases that annotate various brain regions including maps of gene expression (the Allen Brain
Atlas).
How do we have understanding of how individual brain cells function
Understanding how individual brain cells function (membrane potentials, action potentials,
transport of molecules, Hodgkin-Huxley etc) is based on early studies in cephalopods, including
the “giant axon” of the squid, visible to the naked eye
Learning and memory - habituation and sensitisation
Eric Kandel and colleagues tackled complicated issues of learning and memory in a mollusc,
Apylsia, using habituation and sensitization of the gill withdrawal reflex. The neurons
responsible are collected in a ganglion comprised of large and identifiable cells. Sensitization, a
model for “long term” memory formation requires activating immediate early genes (IEGs),
IEGs
ranscription factors that respond rapidly (30 min) and serve as TFs for other TFs.
Uses of TFs
Transcription factors are now used across vertebrates to identify brain regions during the
activation of a behavior. Erich Jarvis and colleagues visualized IEGs in the brain of zebra finches
after singing or hopping on the perch and showed that active pre-motor areas during singing
(HVC and RA) are surrounded by areas active during hopping
Organoids
Organoids can be viewed as a model for human brain development
Gurdon and Yamanaka received the Nobel Prize in 2012 for the discovery that
a) Gurdon:
the nucleus of a “terminally” differentiated cell can give rise to an embryo that is transplanted
into an egg. That embryo can give rise to male and female offspring (i.e. the nucleus can
“direct” the formation of germ cells.
Yamanaka figured out which TFs a cell had to express in order to become pluripotent, narrowed the field from 24 TFs to 4: Oct 2, Sox 2, Kl 4 and c-Myc, first for mouse
fibroblasts and then for human fibroblasts derived from epidermal (skin) cells.
Complexity of cells in tissue culture vs organoid vs brain
Cells in tissue culture, organoids and an actual brain range from simple to highly complex
systems. Within the organoid neurons occupy discrete layers as they do in the cerebral
cortex but there are fewer layers and cell types.
How to make an organoid
Dissociate the cells, culture them in a bioreactor (to increase
access to nutrients and O2), with the 4 Yamanaka factors (best), or agitate them, or plate
them on a scaffold (ALI-COS), on a chip, put two organoids together (assembloid) or implant
them into a mouse brain (creating a chimera).
How to we characterise cells in an organoid
Using single cell sequencing (and “dimensionality reduction”; UMAPs) to characterize cells
in an organoid.
Use of organoids
Organoids can be used to compare gene expression between genetic males and females
(note that no hormones - estrogens or androgens- Cells are cultured are supplied). XX vs XY.
GO terms = gene ontology (i.e. biological functions associated with expression of that gene).
Both XX and XY organoids include cells that express nestin (i.e. are neurons).
Neural disorders that can be addressed using organoids.
Ethical concerns of organoids
Brain organoids from developing forebrain develop optic cups that are sensitive to light.
Neurons from the human transplant extend axons ipsilaterally and contralaterally into
the host mouse cortex.
Huma-derived organoids transplanted into a mouse brain develop spontaneous activity at
frequencies up to 40Hz.
Benefits/drawbacks of human brain organoids
While human brain organoids derived from iPSCs (induced pluripotent stem cells) have many advantages as models for
human-specific features of neural development and (when derived from a patient’s own
epithelial cells) some neurological disorders, connectivity of neurons in cerebral organoids (or assembloids) has not been extensively characterized.
Neural circuits and spinal circuits
Neural circuits (e.g. the spinal circuit supporting the “stretch reflex” are embedded in neural systems (e.g. the motor control system); descending inputs (e.g. brainstem to spinal cord, motor cortex to brainstem) can influence spinal circuits.
What does stretching the muscle (e.g. tapping the knee) do? –> Spinal circuit
Stretching the muscle (tapping the knee with a hammer) is a sensory input conveyed via
neurons in the dorsal root ganglion (Sensory neuron) to motor neurons in the ventral horn of
the spinal cord (extensors or flexors). Activating the inhibitory interneuron (purple) prevents
contraction of the flexor muscle, contraction which would have opposed activation of the
extensor muscle.
How can we record IPSPs and EPSPs
IPSPs and EPSPs can only be recorded with an intracellular electrode while action potentials
can be recorded with both IC and EC electrodes.
Stretch reflex
Jendrassik manœuvre
Brainstem circuits modify spinal reflexes. In the Jendrassik maneuver, descending input from more anterior levels of the CNS amplifies
the reflex.
The manoeuvre reduces activity in normal brain stem descending inhibitory pathways the control spinal reflex neurons, and reducing the inhibition at the spinal level may lead to exaggerated reflex responses (?).
How do you trace long-distance synaptic connections? What does tracing rely on?
Tracing long-distance synaptic connections relies on axonal transport. Anterograde: cell
body to terminal. Retrograde: terminal to cell body. Cargo is also transported from
cell body to dendrites. Both retrograde and anterograde transport occurs along microtubules ( - end towards cell body, + end towards axon terminals)
using molecular motors: kinesin and dynein.
How do we use axonal transport to trace connections
Using axonal transport to trace connections. Retrograde transport of HRP (horseradish
peroxidase, a plant enzyme) from motor neuron terminal in muscle to motor neuron cell body
in spinal cord visualized with a chromogen (TMB) and hydrogen peroxide (the enzyme catlalyzes
the oxidation of the chromogen.
How can you trace circuits>
To trace circuits you need tracers that are transsynaptic (retrograde or anterograde).
Filling DRG neurons (a ganglion contains many neurons) with dye outlines
which DRGs influence spinal cord neurons, including projections directly to the ventral horn and
to the contralateral side of the spinal cord (labelled processes right above the central canal).
How do you trace connections of specific brain regions (brain nuclei) in the CNS?
You can use viruses.
Anterograde tracing (cell body to terminal):
- Statically/monosynaptically restricted –> PHA-L (a lectin virus) or AAV2
- Monosynaptically restricted –> herpesvirus (H129∂TK, thymidine kinase gene required for replication is deleted)
- Polysynaptic –> H129
Retrograde tracing (terminal to cell body):
- Static: CTb, AAV-retro
- Monosynaptic: SAD∂G (EnvA) glycoprotein-deleted ENV-psyeotyped rabis virus.
- Polysynaptic –> pseudorabies virus (PRV)
AAvs
Small, 4.7-kb, ssDNA viruses in the parvovirus family that can infect multiple tissue types. Currently the major circuit tracing tool in mammals.
Retrorade transsynaptic with rabies virus
the rabies virus coat protein can be replaced with a foreign
coat protein (Env) that will recognize and bind to a foreign receptor (TVA).
Rhodopsin expression/activation in mice
Channelrhodopsin and halorhodopsin not native molecules, can be expressed via transfection with a virus or transgenesis.
Can include a cell specific promoters in the viral construct.
Light delivered to the brain activates these manipulated rhodopsins.
How to make a transgenic mouse.
Take the inner cell mass out of a very
young mouse embryo, culture the cells, transfect with a cell marker (could be something
that confers resistance to an antibiotic abnd would be the only one to survive in
vitroinfecgtion, could be a fluorescent protein. Expand the marked cells, treat with K-
factors, put into a new mouse blastocyst, breed chimeric progeny until all cells carry the
transgene.
How to label LLDRG connections in mice
Can use a transgenic mouse in which GFP is expressed in all DRGs; can also just inject a dye.
What are the four circuit architectures
A. Feedforward excitation; B. Feedback and
feedforward inhibition. C. Lateral inhibition D. Mutual inhibition
Architectures of neuronal circuits
Includes back propagating APs, continuous topographic mapping, discrete parallel processing
