Dendritic Spines

Question 1

• Cajal 1888:

Answer 1

Golgi-stained cortex → discovered dendritic spines

Question 2

• Roberts et al (1996):

Answer 2

Post-mortem brains. EM of striatum→ looked at the area of dendritic spines.
o Striatal spines were ~30% smaller than controls in schizophrenics → suggests potentially abberant synaptic conductance/efficiency
o Spines were similar in size in both the caudate and putamen in normal individuals and similar for schizophrenics

Question 3

• Steward and Falk (1985):

Answer 3

Examined DG of developing rat with EM. Over the major period of synaptogenesis.
o Inverse relationship was found between the synapse density and the proportion of spines with polyribosomes.
o Incidence of polyribosome containing spines highest at youngest age and decreased with development .
o Suggest that the polyribosomes represent a structural specialisation of dendrites→ produce proteins involved in synaptic growth?

Question 4

• Steward and Reeves (1988):

Answer 4

Dendritic spines form the DG and the hippocampus of the rat were analysed at EM level in 2 ways
o Ribosomes in dendritic spines
o Interrelationship between polyribosomes, membranous cisterns at the base of spines and spine apparatus→ potentially these organelles may represent different components of a system for the synthesis and processing of proteins related to the synaptic site (like rough ER)
o About 50% of polyribosomes that were present beneath the base of spines were associated with membranous cisterns. However polyribosomes at the head of spines were rarely associated with cisterns
o Polyribosomes are more prevalent during time of synapse growth whereas the spine apparatus are not common until the synapse matures
o Another interesting feature is the presence of spine apparatus: looked a lot like golgi apparatus (membranous cisterns) under the EM . Suggested that may be able to package proteins into organelles and post-translationally modify them.

Question 5

• Emptage et al (1999):

Answer 5

Confocal microscopy to monitor Ca2+ evoked transients in dendritic spines of hippocampal pyramidal cells (CA1 and CA3), hippocampal culture, Ca2+ dye
o AMPA (antagonist CNQX) and NMDARs (d-AP5 antagonist) block synaptically evoked Ca2+ transients
o AMPAR antagonist blocking is relieved by low Mg2+
o Release of Ca2+ transients are mainly due to release of Ca2+ from interna stores, since they are abolished or greatly reduced by CICR antagonists (CPA, a specific blocker of SERCA and RyR)→ But do not depress spine Ca2+ transients generated from bAPs
o Implications for synaptic plasticity as they show that synaptic stimulation can activate NMDAR, evoking substantial Ca2+ release from internal stores in spines without inducing LTP and LTD
o Indicated that CICR is activated by NMDARs

Question 6

• Fischer et al (1998):

Answer 6

Attached GFP to actin (in live cells) in dendritic spines, cultured hippocampal neurons→ observed what happened to levels of actin in living neurons in vitro. Time lapse recordings of living neurons
o Not stationary or fixed in the way people anticipate →Actin constantly moving, undergo rapid dynamic changes
o Revealed large actin-dependant changes in dendritic spine shape (don’t appear to alter the size or density)
o Changes occurred within seconds → anatomical plasticity is rapid
o Spine motility ceased rapidly when treated with drugs that block actin polymerisation and then recovered when the drugs were removed

Question 7

• Jaworshi et al (2009):

Answer 7

EB3 (key microtubule associated protein – tip tracking protein). GFP -Fluorescent microscopy in live cells. Microscopic approaches. Hippocampal neurons
o EB3-GFP to mature hippocampal neurons in culture, live cell imaging → appear and disappear in dendritic spines
o Nocodazole to inhibit MT dynamics → decreased number of mushroom headed spines and increased number of filopodia (reversible effects indicating due to MT dynamics). Also in CA1 region suppressed synaptic potentiation→ link between dynamic MT and synaptic plasticity
o Nocodazole resulted in loss of F-actin from dendritic protrusions whereas expression of EB3-GFP increased F-actin abundance
o Found that dynamic EB3 positive MT plus ends can enter dendritic spines, are required for controlling the levels of F-actin within the spines and are essential for the maintenance of spine morphology and mature synapses
o EB3 appears transiently within dendritic spines and then disappears again→ EB3 enters spines and can modulate spine morphology
o Inhibition of microtubule (dynamics) modulates spine shape via regulation of acting cytoskeleton
o Propose that EB3 labelled growing microtubule ends regulate actin dynamics within dendritic spines → linking dynamic microtubules to spine changes and synaptic plasticity
o Indicates that cargos could be delivered to spines as a function of appearance/absence of microtubules transiently in spines
o Interplay between MTs and actin cytoskeleton

Question 8

• Majewska et al (2000):

Answer 8

Effect of spine motility on calcium dynamics by using 2-photon excitation to photobleach enhanced GFP (eGFP) and image calcium indicators in motile spines Layer 2/3 pyramidal neurons in the mouse visual cortex slices. FRAP = fluorescence recovery after photobleach (stimulating cells)
o Diffusion of EGFP across the spine neck is linearly correlated to the length of the spine neck and is altered during motility. Also find that changes in spine neck length affect the initial fast Ca2+ decay in spines
o (GFP is much larger than Ca2+)
o Over a relatively short period across a number of dendritic spines, the spine length changes quite a bit. → time lapse imaging
o Elongation/retraction of spine neck during spine motility alters the diffusional coupling between spine and dendrite → significantly changes Ca2+ decay kinetics in spines
o Shorter spines have faster recovery than long spines. Significant correlation between spine neck length and the EGFP fluorescent recovery time constant → implies the spine neck length determined the diffusional coupling between spine and dendrite. clearly narrower necks take rather longer to recover. May have important implications for trapping proteins required for synaptic plasticity
o Demonstrate the spines’ ability to compartmentalise Ca2+ is constantly changing

Question 9

• Harnett et al (2012):

Answer 9

Combined 2 photon imaging with Photolytic Glu uncaging → deliver Glu to dendritic spines and use patch electrode recordings on the dendrite to ty and generate a more accurate estimate of synaptic conductance. Measured the amplitude ratio of spine head to parent dendrite voltage across a range of dendritic compartments and calculate the associated spine neck resistance for spines at apical trunk dendrites in rat hippocampal CA1 pyramidal neurons
o The Rneck is large enough to amplify substantially the spine head depolarisation associated with unitary synaptic input by 1.5 to 45 fold, depending on parent dendritic impedance
o Modelling indicated that spines provide a consistently high-impedance input structure throughout the dendritic arborisation.
o Demonstrated that amplification produced by spines encourages electrical interaction among coactive inputs through an Rneck dependant increase in spine head voltage gated conductance activation
o Conclude that the electrical properties of spines promote nonlinear dendritic processing and associated forms of plasticity and storage → enhancing the computational capabilities of neurons
o Amplification increases the activation of v-dependant processes within the spine head, enhances the interaction among coactive spines and increases nonlinear dendritic integration
o Measured resistance that they estimate is rather higher than originally anticipate
o Under these conditions the levels of resistance would influence synaptic currents in a profound way

Question 10

• Bloodgood and Sabatini (2005):

Answer 10

Rat hippocampal neurons pyramidal neurons. Photoactivatable GFP (PAGFP). Photoconvert GFP in dendritic spine → see how long it takes to migrate out. Change the performance of dendritic spines by inducing LTP within to see if the rate constant changes
o Found that spine/dendrite diffusional coupling is heterogeneous
o The size of the spine neck play a very big part in the extent to which there is coupling between the heads of the spines and the dendrites and this turns out is relevant for the corralling/trapping of protein plasticity in species within individual synapses.
o The barrier of diffusion posed by the neck and number of diffusionally isolated spines is bidirectionally regulated by activity of neurons
o Coincident synaptic activation and postsynaptic APs rapidly restrict diffusion across the neck
o The regulation of diffusional coupling provides a possible mechanism for determining the amplitude of postsynaptic potentials and the accumulation of plastic inducing molecules in the spine head
o Incubation with AMPAR antagonist NBQX quicker diffusion, whereas Blockade of GABAAR results in slower diffusion. Altering NMDAR with CPP had no effect
o Reducing activity reduced the fraction of highly diffusionally isolated spines
o Spine neck lengths were reduced after NMDAR and AMPAR blockade
o Looking a bAPs then used NPE-HPTS (photoactivateble)→ pairing EPSP (uncaging glu) and small bursts of bAPs triggered increase in diffusional coupling → therefore restriction of diffusion across spine neck

Question 11

• Yuste et al (1999):

Answer 11

2 photon fluorescence imaging of CA1 pyramidal neurons in rat hippocampal slices. Investigated the mechanisms by which Ca2+ enters individual spines. Different pharmacological and stimulation conditions
o 3 different pathways for Ca2+ influx:→ functional complexity
o High threshold V gated sensitive Ca2+ channels (Ni2+), NMDAR receptors (AP5) and an APV-resistant influx consistent with calcium permeable AMPA or kainate receptors
o . Each pathway is activated under different conditions, with APs opening VSCCs, EPSPs activating NMDA and nonNMDA glutamate receptors, and finally, pairing of APs and EPSPs activating all pathways but with a predominance of NMDA receptors.
o Pathways among different populations of spines and are engaged under different stimulation conditions
o Calcium dynamics of spines are sensitive to the temporal coincidence of the input and output of neurons
o Suggest individual spines are chemical compartments that can perform coincidence detection
o Found supralinearity of AP and EPSP to be NMDA dependant (AP5) and was reduced in the opposite order

Question 12

• Chen et al (2012):

Answer 12

Simultaneously monitor in vivo inhibitory synapse and dendritic spine dynamics across the entire dendritic arbour or pyramidal neurons in adult mammalian cortex → dual-colour 2-photon microscopy. Looked at the inhibitory synapse and dendritic spine remodelling across the entire dendritic arbour of cortical L2/3 cortical neurons in vivo during normal and altered sensory experiences. Monocular deprivation.
o inhibitory synapses on dendritic shafts and spines differ in their distribution across the arbor and in their remodeling kinetics during normal and altered sensory experience.
o inhibitory synapse and dendritic spine remodeling to be spatially clustered and that clustering is influenced by sensory input.
o Our findings provide in vivo evidence for local coordination of inhibitory and excitatory synaptic rearrangements.
o the density of inhibitory spine synapses on apical dendrites increased with distance from the cell soma
o 2 inhibitory synapse populations display distinct temporal responses to visual deprivation→ suggests different involvement in early versus sustained phases of experience dependant plasticity
o Rearrangement of inhibitory synapses and dendritic spines are locally clustered mainly within 10μm of each other and the spatial range of local intracellular signalling mechanism and that clustering is influenced by experience
o Inhibitory inputs could have a profound local influence of performance of signals within a dendritic spine
o Still clusters following MD but the clusters are increased. Fraction of dynamic spines involved in clustered event increases

Question 13

• Sabatini and Svoboda (2000):

Answer 13

Baclofen (GABAb agonist). 2 photon microscopy to image AP induced Ca2+ transients in the spines and dendrites of CA1 pyramidal neurons in rat hippocampal slices
o GABAb differentially modulates bAPs at spines and dendrites, revealing that signalling factors other than Ca2+ (e.g. cAMP) are compartmentalised
o See that metabotropic GABA receptor variant is able to modulate the magnitude of the bAP generated Ca2+ signal in the dendritic spines
o When baclofen is presented the magnitude of Ca2+ signal is reduced in the spine
o Report that each spine contains about 1-20 VSCC, increases with spine volume
o In spines located on the proximal dendritic tree, VSCC normally open with a high probability following dendritic AP
o Activation of GABAb receptors reduced the probability in apical spines to ~0.3 but had no effect on VSCCs in dendrites or basal spines
o Suggest spatial distribution od VSCCs subtypes and their modulatory potential regulated.
o - don’t directly look at bAPs

Question 14

• Padamsey et al (2017)

Answer 14

Hippocampal pyramidal neurons. Lysosome tracker dye throughout dendritic arbour of CA3 and CA1 neurons and in dendritic spines. Various fluorescent imaging techniques
o BAPTA: bAP evoked Ca2+ influxes
o Ca2+ influx through NGCCs triggers lysosomal release (inhibit with Ni2+ and Cd2+)→ could not detect Ca2+ transients under conditions
o bAPs trigger Ca2+ release from lysosomes results in exocytosis of lysosomal protease Cathepsin B → activates MMP-9 (enzyme involved in the ECM remodelling and synaptic plasticity) to maintain activity-dependent spine growth
o Incubate dissociated hippocampal neurons with Magic Red Cathepsin B fluorogenic substrate, which fluoresces upon Cathepsin B-mediated cleavage→ colocalised with LysoTracker → expressed in neuronal lysosomes. When stimulates a loss of both
o Inhibition of either lysosomal Ca2+ signalling or Cathepsin B release prevented the maintenance of dendritic spine growth induced by Hebbian activity → could be rescues by exogenous application of active MMP-9
o ELISA revealed activity-dependant elevations of Cathepsin B in the ECF
o Indicate that activity dependant exocytosis of Cathepsin B from lysosomes regulates the long-term structural plasticity of dendritic spines

Question 15

• Engert and Bonhoeffer (1999)

Answer 15

Combined a local superfusion technique with 2 photon imaging → allowed to see specific regions of postsynaptic dendrites. CA1. Intracelllular recording and calcein (fluorescent dye) Stimulate Schaffer collaterals. Block transmitter release everywhere but small area
o After induction of LTP (but not short lasting) results in new spines on post-synaptic dendrite. In control regions on same region or in slices with LTP blocked, no significant spine growth changes. 2-9 new spines
o Significant correlation between appearance of new spine and increase in synaptic efficiency
o - disappearing spine were seen rarely and in all locations
o - didn’t look at spine volume shape which may also covary with synaptic efficiency
o - no idea if these emerging spines have active synapses . Unlikely at first that the initial increase in strength is due to new spines as they appear no earlier than 30 mins after LTP induction

Question 16

• Hayama et al (2013)

Answer 16

2-colour uncaging of Glutamate and GABA in rat hippocampal CA1 pyramidal neurons. Proximal apical dendrites
o Activity-dependant competition of spines
o Spine enlargement readily induced with repetitive pairing with Glu uncaging and subsequent bAP
o Spine shrinkage never induced by bAP preceding Glu uncaging like expected but was when bAP was paired with GABA uncaging close to the stimulated spine in the LTD
o Shrinkage was blocked by AP5. GABA had to occur within 50ms or preceding bAP
o Applying GABAA agonist muscimol induced shrinkage
o Spine shrinkage and elimination were markedly promoted by the activation of GABAAR shortly prior to AP → This GABA inhibition suppressed bulk increases in cytosolic Ca2+ concentrations but maintained the Ca2+ generated by NMDARs (both necessary for spine shrinkage)
o Unlike spine enlargement, spine shrinkage spread to neighbouring spines and competed with their enlargement (reduced Ca2+ sensitivity)→ process involved the actin-depolymerising factor (ADF/cofilin)
o So GABAergic inhibition directly suppresses local dendritic Ca2+ transients and strongly promotes the competitive selection of dendritic spines
o Spine shrinkage spread to neighbouring spines with LTD protocol except for the specific spine that was exposed to LTP protocol, therefore spine enlargement outcompeted shrinkage without effecting the shrinkage or surrounding spines
o GABA uncaging suppressed the bAP triggered Ca2+ transients
o Demonstrated that both LTP and LTD induced under STDP can be associated with the structural plasticity of spines

Question 17

• Trachtenberg et al. (2002

Answer 17

• ): In vivo chronic time lapse imaging in dendritic spines in barrel cortex (layer 5) in rats over periods of weeks. Altered sensory experience by trimming every other whisker
o Although dendritic structure is stable, some spines appear and disappear.
o Spine lifetimes vary greatly: stable spines, about 50% of the population persist for at least a month whereas the remainder are present for a few days or less. Spine turnover was high but spine density was stable
o Experience dependant plasticity of cortical receptive fields was accompanied by increased synapse turnover→ Suggests that sensory experience drives the formation and elimination of synapses and these changes might underlie adaptive remodelling of neural circuits
o Chess board deprivation of whiskers → considerable effects → increased pool of transient spines present only a single day or less with concomitant decrease in pool of stable spines→ change specific to the deprived cortical areas
o The whiskers that were trimmed had a significantly enhanced surround receptive field compared to the sparde. Way to measure synapse addition and subtraction?
o - Doesn’t directly show the link between RF is due to synapse turnover

Question 18

• Frey and Morris (1997):

Answer 18

synaptic tag hypothesis 2 independent synaptic inputs to the same neuronal population were stimulated in the CA1 region of rat hippocampal slices in vitro. Inhibitor of protein synthesis – anisomycin (translation inhibitor)
o Induction of l-LTP to S1 and S2 were prevented by protein synthesis inhibitors
o We propose that LTP initiates the creation of a short lasting protein-synthesis-independent ‘synaptic tag’ at the potentiated synapse which sequesters the relevant proteins to establish l-LTP
o Show that weak tetanic stimulation (which ordinarily leads to early LTP), or repeated tetanisation in presence of protein-synthesis inhibitors, each result in protein-synthesis dependant late LTP, provided that repeated tetanisation has already been applied at another input to the same population of neurons
o The synaptic tag decays in less than 3 hours
o Indicate persistence of LTP depends not only on local event during its induction but also on prior activity of the neuron
o Proposal that LTP to S1 and then add blocker and then Tetanise S2 → still get lLTP as synthesis at S1 can provide →Prior induction of lLTP in another input, can transform early LTP into l-LTP. Suggests proteins synthesised by LTP in S1 also allowed the induction of the late LTP in S2.
o Critical threshold for tags to be formed?
o – Hanven’t identified a synaptic tag

Question 19

• Bradshaw et al (2003):

Answer 19

Applied protein synthesis inhibitors (emetine- translation inhibiton) to small group of synapses. CA1 pyramidal cells in murine hippocampus
o When applied to apical dendrites l-LTP is impaired but not at basal. If applied to basal dendrites then lLTP is abolished but not apical
o Placing at soma had no effect so can’t be soma delivered
o Thus local protein synthesis modulates the expression of tetanically induced lLTP
o Find evidence that inhibited protein synthesis → however non-selective
o Couldn’t find any ideal reporter that demonstrated unequivocally that they blocked protein synthesis at the local site
o (However more data now to support local synthesis hypothesis)

Question 20

• Nguyen et al (1994):

Answer 20

Transcription inhibitors – antinomycin and DRBRat hippocampal slices.
o See that transcription inhibitors are just as efficient at blocking lLTP than translational inhibitors.
o Blocked Induction of LLTP immediately after tetanisation or drug application of cAMP analogue (cAMP-induced LLTP)
o Requirement for transcription had a critical time window (if application of drug after 2 hours of LTP induction then no effect)
o Late phase of LTP in CA1 region required transcription during a critical period
o So suggests need to generate mRNA to generate LTP for truly long time periods

Question 21

• Sambandan et al. (2017):

Answer 21

MiRNA (regulate gene expression by binding to mRNAs and preventing their translation). Inducible fluorescent probe to explore whether the maturation of miRNA could be regulated in space and time in neurons. Identified a number of potential mRNA targets based on neuropil localised miRNA miR-181a, a neuronal miRNA that is highly expressed in the hippocampus, created a fluorescent probe. Injected into individual hippocampal neurons
o we demonstrate directly that pre-miRNA processing occurs in neuronal dendrites and is stimulated by neural activity at individual synapses, resulting in a local reduction in target protein synthesis→ found that the local maturation of pre-miR-181a, promoted by LFS, led to local downregulation of target CAMKIIα,
o Thus the spatially and temporally regulated maturation of pre-miRNAs can be used to increase the precision and robustness of miRNA-mediated translational repression
o NMDA receptor antagonism completely prevented the stimulation-induced increase in the pre–miR-181a
o Glu uncaging experiments → spatially localised fluorescence increase was observed in the dendritic shaft immediately adjacent to the stimulated one → suggesting generation of mature miRNA. Blocked with AP5
o Used antibody of CAMKII ( a target miR-181a)→ uncaging glutamate and activity dependant maturation of miRNA → In region that that mature Mr181a generated in there was a marked reduction in nascent CaMKII
o Demonstrated that miRNA was spatially localised in an activity dependant way and that activity of individual spines were able to direct specific miRNAs that could be utilised for protein synthesis
o Potentially a mechanism for LTD (but CAMKII activated by HFS → lLTP)