Wiring of the brain

Question 1

Sperry et al 1963

Answer 1

Classic experiments - establishment of synaptic connections regulated by cytochemical affinities that arrise systemically among differently differentiated neurons

Histologically shown that neurons arising from different parts of the retinal select separate paths and have distinct targets in tectum

After optic nerve section, regenerating RGC axons projected back to roughly correct, retinotopically appropriate sites in the optic tectum

However does not prove chemical gradient drives this – tried to eliminate other possible factors (i.e mechanical conditions of growing medium kept same) and concluded must be chemoaffinities.

BUT doesn’t directly prove that it is – no chemical identified, also controlling other factors challenging (how can you control mechanical conditions with medium)

Question 2

Walter et al 1987

Answer 2

Identified molecular activity in membranes isolated from optic tectal cells

Treatment of posterior membrane with high temperature abolished preference of temporal axons to extend neurites on anterior membranes

Heat treatment of anterior had no effect therefore repulsive from posterior membrane

Question 3

Nevin et al 2008

Answer 3

No obvious alterations in the proper laminar targeting of RGC inputs within the superficial layers of the optic tectum in dark-reared compared to control zebrafish

However, dark rearing does not necessarily deprive the visual system of all activity as spontaneous activity may provide surrogate for patterned activity evolutionary for amniotes to have replacement activity when cannot visually interact with environment

Question 4

Benjumeda et al 2013

Answer 4

Silence projections by in-utero electroporation of Kir2.1 (K+ channel which reduces excitability) does not prevent axonal pathfinding or targeting in the SC, but this manipulation does result in less elaborate, more diffusely arborizing axon terminals, indicative of a degraded retinocollicular map

Proves permissive but no evidence it is instructive

Question 5

Richards et al 2010

Answer 5

Receptive field of xenopus larvae examined, expose animal to bias sensory environment (present drifting upward bar)

Reexamine receptive field and compare to normal. The difference in field presents the image shown in training.

Environment influences the connectivity - instructive.

Question 6

Constantine-Paton et al 1989

Answer 6

After several weeks of tectal NMDAR blockade (with APV into tadpoles) a focal injection of a retrograde tracer was made into the optic tectum

Compared with sham treated control animals, the animals that had undergone blockade exhibited a pronounced degradation of input convergence resulting in less precise retinotectal maps

This facilitates a corrective mechanism too

When two such axons from the same eye terminate in the same part of the tectum, Hebbian mechanisms should facilitate their convergence, but in the case where two axons from different eyes attempt to terminate in the same location they may be forced apart by competitive mechanisms as a consequence of their poorly correlated firing patterns - segregation into ocular dominance bands

Question 7

Munz et al 2014

Answer 7

LTD promotes neuron segregation

Vivo time-lapse imaging and electrophysiological analysis of individual retinal ganglion cell (RGC) axons

Visually stimulated either synchronously or asynchronously relative to neighboring inputs in the Xenopus laevis optic tectum.

RGCs stimulated out of synchrony - axons grew and added many new branches (find other region)

Question 8

Belluscuio et al 1999

Answer 8

Co-expression of the marker proteins tau-GFP with selected vomeronasal receptors led, for the first time, to the visualisation of a vomeronasal map

Slit2 added to axon dish, outgrowth occurs everywhere but away from Slit2

Immunostaining on tissue samples with specific antibody - Slit2 expressed at midline of brain

Causes neurons to maintain laterality and prevents crossing at the midline – neurons have robo receptor which binds slit 2

Induce rapid axonal synthesis of proteins that promote actin disassembly such as cofilin and RHOA

Question 9

Lopez-Bendito et al 2006

Answer 9

GFP expressing brains -culture eminence tissue (LGE derived migrate tangentially into the corridor)

Mechanically block ventral migration with semipermeable membrane

Cocultured with thalamocortical axons

If prevent LGE corridor cells from accessing with membrane, there is no bridge for thalamic axons to pass through - no TCA migration to the thalamus

BUT in-vitro experiment - potential lack of natural behaviour and expression of corridor cells - role may not be accurate to that in-vivo during development - may lack other factors that required for TCA migration -corridor cells may just be a compensatory mechanism

Improve - In-vivo model Mash-1 mouse embryos - defect in the early development of the basal telencephalon that correlates with an abnormal pathfinding of TCAs

Potential defect in corridor formation

LGE transplantation - and remarkably noticed that restored growth of WT-TCA into Mash-1 mutant in conduction with corridor formation.

Question 10

Bielle et al 2011

Answer 10

Remove corridor and flipped it

If there are guidance cues one would expect the flip to affect how axons pass through the corridor.

Outgrowing rostral thalamic afferents change course (80% showed changes). Guidance cues must be present

One investigated is Slit1 - in situ hybridisation shows visible in rostral regions of corridor but not present causally

Slit1 -/- axons labelled with Dil anterograde abnormally reached cortex

Question 11

Magnani et al 2010

Answer 11

Mice (Golli-tau-GFP x Pdn) – Gli3 hypomorph as the KO is lethal. Complete absence of cortical subplate cell expression (ventral/dorsal telencephalon disrupted)

Transplantation assay to test the ability of Pdn/Pdn corticofugal axons to cross the PSPB (pallial/subpallial boundary) and enter the striatum.

When we transplanted Pdn/Pdn;GFP+ axons into control GFP- cortex, observed GFP+ axons penetrating the ventral telencephalon

BUT when transplantation of control;GFP+ thalamus into the cortex of Pdn/Pdn;GFP− embryos corticofugal axons do not enter the striatum

Suggesting Pdn/Pdn ventral telencephalon has lost its intrinsic ability to guide corticofugal axons into the striatum.

However, although corridor still forms, Gli3 also affects the corridor (reduced LGE, smaller) - how can we distinguish role - should have control with normal corridor and abnormal telecephalon

Question 12

Garaschuk et al 2000

Answer 12

Large-scale oscillatory calcium waves in the immature cortex.

Imaged slices BUT TWO PHOTON IMAGING allowed us to record at a depth at which we would expect cells to be minimally, if at all, affected by the slicing procedure

Two-photon imaging revealed immature cortex, showed giant depolarising potentials

If block glutamine, ablate GDPs in the cortex BUT in hippocampus they survive

AMPA signalling more prominent in cortex early on

Question 13

Bonifazi et al 2009

Answer 13

Two-photon imaging of hippocampal cells in acute in vitro slices. Detect activity using fluorescent Ca2+ imaging (Fura-2AM)

GAD67-GFP KI mice to selectively identify GABAergic neurons

Hub neurons identified with high connectivity index

Targeted electrophysiological recordings and stimulation of neurons with a known degree of functional connectivity (HC cells) - induced sustained action potential (AP) firing that significantly decreased the occurrence of GDPs

Hub cells synchronise.

Hebbian theory - hub cell starts to fire, strengthens connections , until neonatal activity can cease as connections have formed

Causal influence of HC cells on network dynamics - challenging to conclude as targeted artificially - cannot be certain this would occur in-vivo - also computationally targeted - which cells would in reality

Question 14

Marques-Smith et al 2016

Answer 14

Performed recordings in a transgenic mouse line, Lpar1-EGFP, which labels a population of SPNs - laser scanning photo-stimulation (uncaged glutamate), mapped circuits through development

Weakening of inhibitory interneuron input (thalamorecipeint) from deep layers of the cortex (transient L5b) onto layer four as development progresses and acquisition of thalamocortical synapses - activity dependent

Scaffold not present in Nrg1 overexpressing mice -neuregulin 1 (Nrg1) receptor family has been shown to selectively regulate the formation premature PV+ IN-pyramidal cell synaptic connections through ErbB4 signaling

Drive parvalbumin interneurons to integrate early to outcompete interneurons

This early imbalance in inhibition towards local circuits results in altered sensory input via the thalamus

Schizophrenia model (based on GWAS so good model – however when translate to animals different as smaller subplates)

Limitations is that perhaps too fine scale – spatial resolution at expense of mapping full circuit. Great for mapping an individual whisker barrel (L4) but not viable for entire depth of cortex

Question 15

Van der Loos 1970/3

Answer 15

Golgi stain showed clustering of layer IV neurons – one to one correspondence with whisker pad - barrels

After cauterization of whisker rows in newborn mice
anatomic investigation of the barrel field layout several weeks later revealed striking prevented formation of corresponding barrel structures.

Mice, selectively inbred for abnormal, additional whisker follicles (supernumerary whiskers) at different loci in the whisker pad, corresponding extra barrels formed in the somatosensory cortex

Question 16

Salichon et al 2001

Answer 16

Lack of 5-HT1B rescued the phenotype of MAOA KO mice - 5-HTb mediates the developmental effect of excessive 5-HT

Question 17

Laurent et al 2002

Answer 17

Whole-cell patch-clamp recordings of layer 4 neurons

Significant reductions in EPSPs post application of 5-HT (enhanced with more serotonin)

5-HT1B expressed on thalamocortical axons - In-situ hybridisation of 5-HTb at different developmental stages pinned expression to a limited temporal window (P0-P7 high, disappeared by P14) - act as regulators of thalamocortical development through the inhibition of glutamate release

TC axons autoregulate their glutamate release via extracellular 5-HT binding to their 5-HT1B receptors

BUT ignores the physiological conditions of 5-HT release by applying it - not reflective of in-vivo dynamics

Question 18

Protocortex O’Leary et al 1991

Answer 18

Grafting experiments showing that pieces of visual cortex transplanted onto somatosensory cortex could develop TC axon patches

Therefore cannot be an intrinsic property?

Ignores the concept that differential location through a developmental period may influence the characteristic of that tissue through other local factors and neighboring tissue (not necessarily in response to TC axon)

Question 19

Protomap Fukichi-Shimogori & Grove 2001

Answer 19

Ectopically express Fgf8

Increasing levels of Fgf8 at the anterior pole of one hemisphere (achieved by in utero electroporation), results in cortical arealisation defects, which lead to caudally misplaced thalamic axon terminations

Barrell regions are moved around - they are misplaced but maintain their function

Question 20

Lake et al 2018

Answer 20

New frontier - high-throughput methods for single-nucleus droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive site sequencing (scTHS-seq).

Acquire nuclear transcriptomic and DNA accessibility maps - epigenetics

Question 21

Alzubi et al 2017

Answer 21

HOWEVER IN humans large overlap between these two TFs in sensory areas, while in mice this was not the case

Although this may reflect higher resolution in human sequencing studies

Perhaps the expansion of cortical COUP-TFII expressing territory in human fetal brain, overlap with SP8 mirrors the increased size and complexity of the association areas of the ventro-temporal cortex -allows for association between sensory and motor areas, with them interconnecting.

Question 22

Terakawa et al 2013

Answer 22

Cadherins (adhesion molecules) differentially expressed in cortical areas by late development,

Cad6 expression in somatosensory and auditory cortex as well as the thalamic nuclei innervating them

Importantly these expression patterns can still be perceived in earlier developing cortex prior to thalamic innervation (protomap)

However differences between patterns of cadherin expression in both postnatal and embryonic cortex differ between marmoset and mouse - important to study the expression of these molecules in human in detail in the future

Question 23

Reillo et al 2011

Answer 23

Experimental closure of the eye (“enucleation”) in ferrets during early postnatal development results in reduced proliferation of OSVZ radial glia and a reduction of both dLGN and primary visual cortex size

Question 24

Vasung et al 2020

Answer 24

In order to determine regional volumes of cortical compartments (cortical plate (CP) or subplate (SP)) and estimate their growth rates, we acquired T2-weighted in utero MRIs of 40 healthy fetuses and grouped them into early (<25.5 GW), mid- (25.5–31.6 GW), and late (>31.6 GW) prenatal periods.

No significant difference was found between periods in regional volume fractions of the CP or SP.

However, during the early and mid-prenatal periods, we found significant differences in relative growth rates (% increase per GW) between regions of cortical compartments.

Thus, the relative size of these regions are most likely conserved and determined early during development whereas more subtle growth differences between regions are fine-tuned later, during periods of peak thalamocortical growth.

This is in agreement with both the protomap and protocortex hypothesis.

BUT size may not correlate with regional specification, combine with single cell sequencing perhaps. Non-invasive means of sampling tissue

Question 25

Bae et al 2014

Answer 25

Deletion mutation in a regulatory element of GPR56 that causes intellectual disability and epilepsy, and selectively disrupts human cortex surrounding Broca’s area.

GPR56 encodes a receptor required for normal cortical development that regulates progenitor proliferation.

GPR56 splice forms are highly variable between mice and humans, and the disrupted regulatory element only affects gyrencephalic mammals

This study provides a mechanism by which controlling regional GPR56 expression by multiple alternative promoters could influence evolution of cortical arealisation by control of stem cell proliferation and gyral patterning.

Question 26

Kriks et al 2011

Answer 26

Neurons from human PSCs generally show poor in vivo performance.

Novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells.

Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling.

In vivo survival and function is demonstrated in Parkinson’s disease models using three host species. Scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth

Question 27

Lui et al 2004

Answer 27

Using reverse transcription-PCR, in situ hybridization, relative contributions of two subtypes of NMDA receptor (NR2A, NR2B) were measured from p2-p15.

Quantitative switch in the dominant synaptic subunit from NR2B to NR2A. Change in the cellular expression of NR2A

Coupled to shortening of NMDA mediated EPSCs during critical period of postnatal development - narrower correlation window, finetuned plasticity

No dynamic insight - snapshots of expression -calcium imaging

Gambrill et al 2011: fluorescence microscopy Ca2+ imaging - longer duration of NR2B results in greater Ca2+ entry through NMDA and long term plasticity promotes stabilization of circuitry

Question 28

Connor et al 2011

Answer 28

Schizophrenia is a deficit in transient cortical circuits

In schizophrenia - greater proportion of interstitial white matter cells (remnant of the subplate cells) especially in the frontal area

Cells don’t get removed in the correct proportions.

Question 29

Mirnics et al 2000

Answer 29

Schizophrenia as a presynaptic secretory machinery deficit.

Synaptic abnormalities seen histologically - drastic reduction in the synapses

Abnormal pruning in schizophrenia - drastic synapse reduction

As a result of disruption in genes coding for presynaptic proteins and regulator of G-protein signaling 4 (RGS4) function - common polymorphism

Question 30

Fish et al 2006

Answer 30

RNA interference in telencephalic NE cells, mitotic spindle poles lack Aspm protein, and the cleavage plane of NE cells is altered - undergo asymmetric division, (i.e. apical plasma membrane is inherited by only one of the daughter cells).

Reduction in the number of NE progenitor cells upon Aspm knock-down relative to control.

Primary microcephaly observed in humans with mutations in ASPM.

RNAi - off target affects are likely

Question 31

Rakic 2013 theory of brain size

Answer 31

• The absolute increase in surface area and number of cortical neurons combined with the selective expansion of frontal and parieto-temporal cortical regions is thought to underlie the emergence of advanced cognitive abilities in humans, including perception, language, and reasoning

Question 32

Li et al 2017

Answer 32

Negatively regulated also -PTEN-AKT cascade regulates growth

Megancephaly associated with PTEN mutation

3D organoid system with PTEN homozygous mutation (increased AKT activity) (crispr KO)

Cell cycle re-entry, and transiently delayed neuronal differentiation – MORE ki67+ cells confirmed with qt-pcr, resulting in a marked expansion of the radial glia and intermediate progenitor population - increased size and folding (fluoresence microscope)

Treated developing PTEN mutant human organoids with GDC-0068 or MK-2206, known inhibitors of AKT activation - reduced phenotypes suggest AKT mediated effect

Organoid system lacks cellular structure and communication of in-vivo model - overcomes lack of gyri in mouse brain.

Question 33

Apoptosis in brain size?

Answer 33

Using terminal dUTP nick end labelling (TUNEL) (DNA laddering characteristic of apoptosis) to detect dying cells have suggested that there is virtually no cell death during telencephalon development

Contrary to these findings, reported unexpectedly high rates of embryonic cell death using the in situ end labelling + (ISEL+) (fragmented DNA) technique

MORE sensitive than TUNEL however results may depend on how the samples were taken - takes a snapshot of activity

Annexin V-based assay is one of the most sensitive techniques to detect exposure of phosphatidylserine (PS) residues in the outer membrane of cells - couple to YFP with genetics

Question 34

Rakic 2008 Radial unit hypothesis of neocortical expansion

Answer 34

1000 fold increase in cortical surface without a comparable increase in its thickness

Increase in the number of radial columnar units without significant changes to neuron number

Question 35

Meyer et al 2018

Answer 35

Second wave of cajal retzuis (‘adult’ neurogenesis)- contributes to tangential migration

Calretinin and GAD expressed in subpial granular layer - immunohistochemistry foetal brains

Long-distance tangential migration may be crucial for human interneurons which have to travel over increasingly longer distances as the cortex grows.

Fewer caudate neurons (new) in the autistic brain - calretinin positive interneurons migrate laterally

Question 36

Rakic migration

Answer 36

Serial-section electron microscopy - late stages of primate cortical development (cerebral wall thickness > 3,000 μm) - the entire length of a migrating neuron maintains an intimate apposition to radial glial fibres (cerebral wall is considerably thicker - radial glia act as a scaffold)

Question 37

Chai et al 2016

Answer 37

Reeler mice - migration defect - highly disorganised cerebral cortex - outside first inside last KO Lissencephaly with cerebellar hypoplasia
(observed by real time microscopy and histology)

Rescued by overexpression of coffin (actin-depolymerising protein downstream of reelin signalling pathway)

NOT significantly difference in coffin overexpression alone vs reeler+ coffin – but both were greater than the control significantly. perhaps beneficial to have overexpression regardless of initial phenotype

Question 38

Henver et al 2006

Answer 38

Sequential expression of combinatorial transcription code

E.g. Pax6, Tbr2, NeuroD, Tbr1 expressed as cell migrates further, induces change from radial cell to immature neuron.

Question 39

Kwan et al 2008

Answer 39

SOX5 – expressed in the subplate cross suppresses expression in other layers

Fezf2 and Bcl11b, transiently expressed in all subtypes of newly post-migratory early-born neurons, are subsequently downregulated in layer 6 and subplate neurons, thereby establishing their layer 5-enriched postnatal patterns.

In Sox5-null mice, this downregulation is disrupted, and layer 6 and subplate neurons maintain an immature differentiation state, abnormally expressing these genes postnatally.

Chromatin immunoprecipitation (ChIP) - anti-SOX5 antibodies specifically precipitated this DNA in Fezf2 but not KO mice, confirming in vivo binding of SOX5

Small sample size

Question 40

T Graham Brown (1911)

Answer 40

Experiments show that you could generate rhythmic behaviour could be seen in absence of sensory feedback

Question 41

Bucher et al (2007)

Answer 41

The stomatogastric ganglion (STG) of the lobster has only ~30 large neurons

The neurons are accessible and easy to record from (prior to advent of patch clamp) - examined in isolated can continue to produce rhythmic motor patterns - movements then produced

However - with such a simplistic CNS surely centralized system would be observed in these species -does not necessarily give us insight into mammalian systems

Question 42

Khein et al 1996

Answer 42

Microdissection techniques

Cut away dorsal cord and found that there was still locomotor activity (CPG ventral)

Cut commissure between the LEFT and RIGHT: drifting was seen, right and left phasing was uncoordinated, (so CPG is bilateral and coordination is needed)

Question 43

Butt et al (2002)

Answer 43

Cut spinal cord - stimulate L4/5

Insert patch electrode in contralateral spinal cord and motor neurons on the other side

Record from commissural neuron – overserve if there is antidromic action potential (prove connection)

Now cells identified - their rhythmicity was analysed, some showed rhythmicity

BUT no two neurons behaved the same - huge diversity was seen (of roughly 90 cells that were analysed) . Even in a simple behaviour there is a diverse range of cells

Mammalian CIN system is more complex than lower vertebrates

Question 44

Briscoe et al 2000

Answer 44

In-situ hybridisation shows differential Shh expression along axis generated TF gradient

Explants exposed to Shh concentrations

Repression of Irx3 required ∼3 nM Shh, a concentration greater than that required for repression of Pax7, Dbx1, and Dbx2 expression, but less than that required for complete repression of Pax6.

Question 45

Britz O et al. (2015)

Answer 45

Heterogeneity using tripartite genetic system

En1 and Gata3 selectively mark V1 and V2b

Tripartite intersectional genetic system (Cre and FlpO) - selectively express the diphtheria toxin receptor in V1 or V2b INs that are located in the caudal spinal cord - selectively sensitive to diphtheria toxin (DTX).

Upon selective gene disturbance with diphtheria toxin, V1 interruption led to extreme flexing of the hindlimbs, while V2b interruption caused exaggeration of hindlimb extension

Criticism: by killing one cell type might cause defecits in others - complete circuit failure?

Question 46

Bikoff et al. (2016)

Answer 46

Interneurons were isolated by fluorescence-activated cell sorting (FACS) from spinal cords

We estimated the number of neuronal types within the parental V1 population through application of a Bayesian sparse linear regression algorithm that assigns V1 interneuron cell types on the basis of TF expression and settling position

Analysis assigns 50 ± 2 V1 interneuron cell types, some defined by as many as nine TFs

BUT taking at a single time slot in development - we do not know how dynamic expression is

Question 47

Kishoreet al 2014

Answer 47

Zebrafish behavioural studies
Voltage-clamp recordings from motoneurons

As swim frequency increases and larger, lower Rin motoneurons are recruited, increase in excitatory drive to these cells.

Inhibition increases relatively uniformly to all motoneurons,
BUT at faster swimming speeds, high-Rin motoneurons in-phase inhibition dominates anti-phase inhibition.

Hybrid model of motoneuron recruitment during locomotion - different pools are recruited.

Question 48

Alilain et al 2008

Answer 48

Transducing spinal neurons in and around the phrenic motor pool to express ChR2

Light activation of ChR2-expressing animals was sufficient to bring about recovery of respiratory motor function in cervical SCI

Robust rhythmic activity persisted long after photostimulation had ceased.

This recovery was accomplished through a form of respiratory plasticity and spinal adaptation which is (NMDA receptor dependent - NMDA receptor antagonist MK-801 eliminated cycling of increasing diaphragmatic EMG activity after photostimulation).

HOWEVER circuity controlling breathing far more simplistic

Question 49

Somogyi et al (1995)

Answer 49

Synchronization of neuronal activity in hippocampus by individual GABAergic interneurons:

Record electrophysiologically pyramidal cells randomly firing, then rebound of synchronous firing

Single interneuron firing synchronizes the pyramidal cells - spatial summation and thus information transfer. Interneurons direct brain function by encouraging spatial summation

Question 50

Nery et al 2002

Answer 50

Lineage tracing studies - transplanted tissue LGE/MGE expressing alkaline phosphatase into WT animals

Animals where then killed and slices were imaged cortical interneurons present in cortex - in-situ hybridisation identified these cells to have GABA interneuron markers

Regardless of transplantation site

Question 51

Butt et al 2005

Answer 51

Cut out ganglionic eminence tissue from GFP mouse

Transplant into WT animals (green MGE in WT)

Mice were allowed to grow up and experimented

Different cells were found from the MGE vs CGE

MGE cells - Interneurons targeting the cell body and proximal dendrites

CGE cells - Interneurons targeting distal dendrites

Question 52

Butt et al 2009 compensation

Answer 52

KO reduces MGE. KO also makes lungs non-functional - leads to animal death

SO use Cre inducible expression

Dose with tamoxifen - mosaic deletion BUT coupled to GFP so can tell which cells KO

(MGE) soma targeting cells are removed and replaced with green fated dendrite targeting population from CGE