Hmb360 Lec 11 Rna And Neuronal Transcriptomes

Front 1

1) Describe the classical view and interest within neuroscience.

s6

Back 1

1) -> Neurons identified by their electrophysiological and morphological properties based on previous methods.

-> Connectivity and molecular features of neurons

-> Combine these features to characterize neuronal diversity

Front 2

1) What method can be generally used to characterize neurons?

s7

Back 2

1) -> Whole-cell patch-clamp recordings on brain slices.

-> Followed by backfilling of the recorded cells with inert dyes for morphological characterization.

-> This allows for aspiration of soma and single cell RNA-seq of the aspirate (backflow) to determine transcriptome of the identified neuron

Front 3

1) Describe the whole-cell patch clamp method, and what can be yielded from it.

2) Describe the change in fluorescence during the patch clamp method

s8

Back 3

1) Whole cell electrode placed on neuron, makes contact with the neuron, backfilled with a dye. As breaks through membrane of neuron, dye will get into cell.

The electrode has high resistance seal (gigaohm seal).

Once looked at morphology of the cell through recording the backflow, can suck on the electrode and you can harvest the cytoplasm content.

The content includes many factors including RNA transcripts.

2) The fluorescence would decrease as the content is harvested.

Front 4

1) What can be understood from transcriptome analysis?

2) What methods have previously been used for transcriptome analysis? What have new developments allowed us to learn?

s9

Back 4

1) There can be non-homogenous cells even within small patches. Can identify subclasses of neurons based on how they connect with each other and how they electrical activity works within their neuronal network.

2) past: candidate marker analysis by mixture of patch-clamp analysis and single-cell semiquantitative PCR (qPCR)

new: allowed ID of novel cell types with distinct mechanisms

Front 5

1) What are some important molecular classifications of neurons done in previous RNA-seq experiments.

2) What are current challenges and what work is being done?

s10,11

Back 5

1) neurons in somatosensory cortex and CA1 subfield of the hippocampus into 47 subtypes, including 16 subclasses of interneurons.

2) even with work using microarrays, multiplexed qPCR and even proof of concept RNA-seq on single neurons, no robust method existed to study patch clamp electrophysiology, morphology (post hoc morphological reconstructions) and transcriptome profiles of the same neuron.

Front 6

1) Describe the experiment done by Qui et. al and the results they got. Describe the general overview of how the experiment done in detail.

s12-13

Back 6

1) -> attempted RNA seq on 3 neurons through patch pipette without prior patch clamp recordings.

-> They performed on brain slices, more difficult.

-> Data had poor quality, no functionality to go along with it. Didn't have electrophysiological recordings, couldnt analyze firing patterns, etc.

Just had gotten RNA

Methods:

-> Looked at interneurons from somatosensory cortex. When have interneuron gene and specific subclass of interneuron, they see both colours.

-> Aspirations: Have electrode touch membrane and record membrane resistance. Increase voltage inside pipette (square wave positive pulses) since RNA is negative. Kept negative pressure to suck cytoplasm.

-> Less than half of a drop, transfered to PCR reaction tube and do amplfication, do RT and convert RNA to cDNA. Can then do single cell RNA sequencing.

Front 7

1) How is Patch-Seq transcriptome used afterwards?

s14

Back 7

1) Align Patch-Seq transcriptomes with larger single-cell data to achieve higher quality classification, to classify cortical interneuron types thought to be homogenous

-> Produce quantitative data set that resolves mRNA for all ion channels, receptors and synaptic proteins at the same time.

-> Meet prediction and validity of the model using pharmacology based experiments.

Front 8

1) Describe the characteristics of Cholecystokinin (CCK) - containing (+) GABAergic interneurons and why are we interested in them?

s15

Back 8

1) -> Mutually exclusive with parvalbumin neurons. If have CCK, no parvalbumin.

-> Morpholigcal and molecular features thought to form quasi-continuum from axon-targeting to dendrite-targeting interneurons in cortical areas and they may modulate various neuronal functions.

-> They are the plastic and dynamic gatekeepers of neuronal circuits

-> inactivation shown to contribute to anxiety, mood disorders and schizo in animal models

Front 9

1) How can they use confocal photomicrograph for CCK identification?

s16

Back 9

1) GAD67 encodes for glutamic acid decarboxylase, part of the GABAergic pathway; so we know its a GABA neuron.

-> Crosses it with a mice that contains CCK.

-> 2 reporters: Red reporter (for CCK expression in cortical pyramidal cells), green GFP for all sorts of interneurons (Some have CCK)

Front 10

1) During combined electrophysiological and RNA-seq analysis, how many neurons identified as inhibitory and how many as excitatory? What properties did they analyze after?

s17

Back 10

1) -> Around a 83 cells of the somatosensory slice were able to be identified through eletrophysiological/morphological properties and were able to take out the RNA.

-> 45 found to be CCK+. They identified these as inter-inhibitory neurons (I-types)

-> The other 38 neurons they identified as excitatory pyrimidal neurons e.g. didnt have GAD67, GABA markers, etc.

-> For each neuron, the location (cortical layer) and passive (inject current and see result in change in memrane AP) and active membrane (stimulate AP, look at how quickly it goes to max potential, how quickly comes back down, how much it hyperpolarizes) properties were analyzed

Front 11

1) Based on looking at overall staining how many groups of neurons would be identified based on overall pattern of postsynaptic target innervation?

s18

Back 11

1) This is a step further than traditional neuron classifications

-> axo-axonic

-> perisomatic

-> dendrite-targetting cells

Front 12

1) How many subtypes were the neurons classified in, based on patch-clamp electrophysiology?

s18

Back 12

1) 5 types based on electrophysiological properties

-> e.g. Type 1 had properties like smallest AP amplitude, largest hyperpol, most hyperpol

Front 13

1) What did they analyze through Post hoc morphological reconstruction (filling with dye and analyzing backflow)? What did they observe in Type 1 interneurons?

s19

Back 13

1) Used to identify the axonal nests (how axons form around themselves) and dendritic arbors for the 5 subtypes.

-> Saw differences in cell morphology, in addition to elctrophys features as seen before

-> e.g. type 1 interneurons had equally sized processes throughout e.g. axons/dendrites as observed through light microscopy

-> type 1 interneurons maintained gap junctions with neighbouring nonpyrimidal cell and to L2/3 pyrimidal cells, when used Lucifer yellow dye

Front 14

1) So what results did they observe when they pulled out the RNA and did seq?

s21

Back 14

1) Saw different transcripts at different levels. A large list of differences.

e.g. type 1 and type 2 dont express vasointestinal peptide like in type 3.

Front 15

1) Describe how the RNA suction was done (in more detail than mentioned before)

2) What results did they get following RNA seq? Success?

s22

Back 15

1) -> apply continuum of square wave voltage pulses (+20 mV from holding potential -5 mV, each 5 ms in length, every 5 ms intervals) to capture negative RNA

-> 0.8 - 0.9 μl of sample ejected into 0.6 μl of lysis buffer, then RNA seq performed through RT

2) 1.6 million reads per cell, but only 40% mapped uniquely to ~2000 distinct genes (using UCSC Genome Browser conservative gene models, mainly protein coding ones)

-> observed around 6000 mRNAs

Front 16

1) What were Pan-neuronal markers to confirm neuronal results?

s24

Back 16

1) genes like Thy1 and Stmn2 were in 79% and 87% of the cells. -> Confirmed neurons generally.

-> I type neurons expressing Gad1 gene (encoding GAD67) and for Gad2 (encoding GAD65) as well as CCK

-> Most cells expressed these. Confirmed inhibitory neurons.

-> the cells also contained mRNA for vesicular inhibitory amino acid transporter and lacked vesicular glutatamate transporter

Front 17

1) What others stats confirmed the inhibitory nature of the neurons?

s25

Back 17

1) -> Expressed mRNAs for neuropeptides (e.g. Vip) and Ca2+ binding proteins (Calb1), markers known to co-exist with CCK in interneurons.

-> none of the cells contained parvalbumin mRNA.

Front 18

1) What did molecular analysis show within the I-type interneurons?

s27

Back 18

1) They quantitatively measured expression of channels, ion pumps and receptors.

-> Patch clamp allowed for measuring difference of any subunit detected within membrane AP: they saw change in expression of Na+/K+ ATPase channels.

Front 19

1) What results added a further level of complexity in various subtypes of the I-type neurons?

s29

Back 19

1) -> They checked for expression of type 1 cannabinoid receptor (Cnr1) in RNA-seq, which reported to have highest levels in CCK+ interneurons.

-> but they found that low expression of Cnr1 mRNA in the I type-1, contrasting with 2-5 subclasses that expressed alot more

Front 20

1) What did they observed in orthogonal image stack when they compared I-type 1 and 4?

s31

Back 20

1) -> They compared how they connected with other neurons.

-> Many of the Htr receptors expressed in type 4 were not expressed in type 1.

-> When inject dye into type 1, dye went to other nearby cells, through gap junctions.

Front 21

1) What were the results of applying 5-HT to the type 1 and type 4 interneurons?

s32,33

Back 21

1) Added 5-HT (serotonin) to the bath containing all of the cells, and generally showed depolarization within both types.

-> Then they microinjected directly into interneuron somata (made sure of equal 5-HT load by coapplying a fluorescent tracer)

-> application to type 1 neuron did not cause depol, type 4 interneurons did (they have serotonin ht receptors). RNA seq correctly predicted that type 1 did not have any metabotropic or ionotropic 5-HT receptors.

Front 22

1) What step could be taken to understand the behaviour of the type 1 neurons?

2) What is the next step to follow up the experiment?

s33,34

Back 22

1) morphological reconstruction of the sampled interneurons may explain of type 1 cells in the bathe application.Type 1 cells may have acted as passive followers of network depol at suprathreshold 5-HT load, synchronizing membrane potential with neighboring neurons through gap junctions.

2) Analysis of single cell proteomes. Challenging to do so.

Front 23

1) How do epigenetic tags survive?

s36

Back 23

1) -> epigenetic mods are dynamic and short lived

-> different organisms dedicate some mechanisms to enable passing some tags

-> Some epigenetic responses passed on despite reprogramming

-> Unknown conditions leading to passing of epigenetic memories

Front 24

1) What was discovered as epigenetic tags in the C elegans model? What theory came up?

s37

Back 24

1) RNA silencing spreads across the worm's tissues and even from soma to germline.

Later shown that in certain cases RNAi mediated changes could last for generations.

Theory: Both exogenously derived small interfering RNA (exo-siRNA) and endogenous small RNAs e.g. endo-siRNAs and PIWI-interacting small RNAs (piRNAs) can trigger heritable RNAi