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1
Q

How much people are affected by MDD

A

Major depressive disorder (MDD) affects approximately 17 percent of the population,

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2
Q

What is predicted in the year 2023 for MDD

A

it is predicted to be the number two cause of illness worldwide by the year 20203 .

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3
Q

Symptoms of MDD and what does it implicate

A
  1. cognitive impairment
  2. loss of memory,
    implicating synaptic dysfunction in the pathophysiology of MDD.
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4
Q

What are the 3 molecular and cellular changes found in mDD patients and how were they discovered and what does all these studies demonstrate

A
  1. a reduction of dendritic spine number and function of neurons in PFC in animal models of depression
  2. Postmortem studies also report morphometric changes in PFC of MDD subjects, including a reduction in neuronal body size
  3. This is also consistent with brain imaging studies reporting a reduction in the volume of PFC subregions in MDD subjects

Together, these studies suggest a loss of spine number and dendritic arbor although there is no direct evidence demonstrating this type of neuronal atrophy in the brains of MDD subjects

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5
Q

How were the mechanisms of neuronal atrophy and reduced volume of PFC studied in this study

A

To gain insight into the mechanisms that underlie neuronal atrophy and reduced volume of PFC, we have re-analyzed the data from a prior microarray gene expression study that we conducted of the dlPFC (Brodmann area 9) of MDD subjects and matched controls

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6
Q

What did this study looking at mechanisms of neuronal atrophy and reduced volume of PFC find

A
  1. We classified the microarray data with a 5% FDR11and found that about 30% of the downregulated genes in the MDD group could be related to some aspect of synaptic function.
  2. A more extended range (20% cutoff) identified an additional subset of down-regulated synaptic function-related genes in dlPFC of MDD brains
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7
Q

What synaptic categories were found in this study

A
  1. Regulation of synaptic vesicles
    - [calmodulin 2, synapsins I and III, Rab3A, amphiphysin, and synaptogyrin 3]
  2. Regulation of synaptic strength [neurogranin]
  3. Dendritic spine formation [Rab4B]18, and axonal outgrowth and regeneration [tubulins]
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8
Q

What gene alterations have genome-wide expression studies found of MDD

A

gene alterations of similar functional categories, including 1. cytoarchitecture

  1. vesicular transport,
  2. synaptic transmission,
  3. some of the same synapse-related genes or isoforms of the genes identified here (e.g., subtypes of amphiphysin, synaptogyrin, synapsin, and the Ras superfamily
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9
Q

What did PCR analysis show

A

 PCR analysis demonstrated significant decreases for 5 of the 10 genes, and trends for all but one (amphiphysin) of the remaining genes in the dlPFC of MDD subjects (Table 1).

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10
Q

What did In situ hybridization analysis confirm

A

1In situ hybridization analysis of the five confirmed genes demonstrates enriched expression in gray matter of dlPFC with a laminar distribution in the middle (synapsin I) or middle and deep layers (calmodulin 2, Rab3A, Rab4B and β-tubulin 4) of dlPFC.

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11
Q

What did Quantitative analysis confirm

A

Quantitative analysis confirms that levels of these five genes are significantly decreased in MDD subjects compared to controls

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12
Q

What does CUS stand for

A

chronic unpredictable stress

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13
Q

What did Studies in rodents with chronic unpredictable stress (CUS) show?

A

Studies in rodents demonstrate that chronic unpredictable stress (CUS), considered one of the most valid rodent models of depression, decreases the expression of synapsin I, calmodulin 2, Rab3A, and Rab4B, but not beta-tubulin 4, in the PFC (Supplementary Fig. 1), suggesting that the decreased levels of these synapse-related genes in MDD result from chronic stress exposure that could contribute to depressive behaviors.

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14
Q

How was dendritic morphology examined

A

microtubule-associated protein 2 (MAP2) immunohistochemistry

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15
Q

What did the examination of dendritic morphology show and how was it confirmed

A

evealed decreased staining of dendritic processes in dlPFC layers III through V of MDD subjects relative to controls, which was confirmed by MAP2 immunoblotting of dlPFC micro-punches

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16
Q

What did electron microscopic stereological analysis show

A

Using electron microscopic stereological analysis, a marked decrease in spine synapse number was observed in MDD subjects compared to controls (Fig. 1h).

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17
Q

What did Co-factor analysis reveale

A

no significant effects of medication status, age of first episode age of first episode (<40 vs. ≥40 years old), or suicide on synapse number

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18
Q

What did Examination of the transcription factor binding motifs show

A

Examination of the transcription factor binding motifs in the promoter regions of the decreased synapse-related genes identified 3,266 upstream regulatory elements for 218 transcription factors (TRANSFAC scoring matrix,

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19
Q

12 transcription factor binding sites are localised to where?

A

Twelve transcription factor-binding sites are localized to the upstream regulatory domain of all of the MDD and CUS-altered synapse-related genes (Fig. 2a).

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20
Q

The rat homologues of these synapse-related genes included what

A

 The rat homologues of these synapse-related genes include eight of the twelve transcription factor regulatory elements .

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21
Q

What did Analysis of the microarray data reveal

A

Analysis of the microarray data revealed that one of these transcription factors GATA1 is significantly increased in MDD patients

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22
Q

which transcriptions factors were either not significantly changed in MDD

A

Other transcriptions factors were either not significantly changed in MDD (GKLF and KID31.0- and 1.1-fold, respectively) or were not included in the microarray gene set.

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23
Q

Levels of what GATA isoforms were not altered in MDD compared to controls

A

Levels of the related GATA-2, 3, and 4 isoforms were not significantly altered in MDD compared to controls.

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24
Q

Co-factor analysis revealed what in GATA1 expression

A

Co-factor analysis revealed no effect of medication status on GATA1 expression, and no significant differences between all MDD subjects and groups categorized by age of first episode, number of episodes, and suicide, although the number of subjects per subgroup was small

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25
Q

Studies in the CUS rodent model of depression demonstrate what

A

Studies in the CUS rodent model of depression demonstrate increased Gata1 expression in the PFC, which was completely reversed by chronic administration of fluoxetine (Fig. 2c).

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26
Q

what did anti-depressants for GATA1 MDD and rodents show and why was the difference

A

The ability of antidepressant treatment to normalize Gata1 expression in rodents but not in MDD could be due to the 1. small number of the medicated and un-medicated subgroups

  1. treatment resistance
  2. heterogeneity of the subjects.
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27
Q

what was used to confirm the binding activity of Gata1 to the promoter of the synapse related genes

A

Confirmed by chromatin immunoprecipitation (ChIP) with a Gata1 antibody followed by PCR for the Gata1 binding region of each gene

 Initial studies focused on Rab4B because this class of small GTP-binding protein is required for endosomal recycling that is critical for maintenance of spine size18 and because RAB4B showed the greatest reduction (Table 1).

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28
Q

what did GATA1 antibody chip result in for most genes

A

For most of the genes, Gata1 antibody ChIP resulted in an enrichment of the promoter compared to mock, control ChIP.

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29
Q

How was the possibility that elevated GATA1 underlies decreased expression of the synapse-related genes and the atrophy of dendritic processes examined

A

primary neuronal cultures.

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30
Q

What did Expression of a GFP-tagged rAAV-GATA1 vector in cultured cortical neurons do

A

Significantly decreased the expression of Rab4b

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31
Q

What is FOG1 and what is it regulated by and what was shown

A

Expression of a cofactor target gene, FOG1, that is positively regulated by GATA1 was significantly increased by viral expression of GATA1

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32
Q

how was dendrite morphology analyzed

A

To analyze dendrite morphology, neurons were fixed and labeled with anti-GFP, as well as antiMAP2 antibodies

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33
Q

What did Viral expression of GATA1 show and how was it confirmed

A

Viral expression of GATA1 decreased the complexity of the dendritic arbor the number of spines , and the intensity of MAP2 staining , which was confirmed by western blot analysis

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34
Q

what did Sholl analysis demonstrate

A

Sholl analysis demonstrated that viral expression of GATA1 significantly decreased the number of dendrite intersections, indicating decreased complexity

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35
Q

how was the effects of GATA1 expression on behavior in rodent models of depression examined

A

Control or GATA1 viral vectors were infused into the PFC and the expression and location were confirmed by GFP expression

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36
Q

what did infusion of rAAVGATA1 show

A

Infusion of rAAVGATA1 produced depressive-like behaviors in two established rodent models.

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37
Q

What was found in the forced swim test with infusion of rAAV-GATA1

A

 In the forced swim test, rAAV-GATA1 increased the time spent immobile, a measure of behavioral despair that is reversed by antidepressant treatment

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38
Q

what was found in the earned helplessness model,

A

In the learned helplessness model, exposing animals to inescapable stress causes escape deficits that are reversed by antidepressant treatment.

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39
Q

during the initial block what did infusions of rAAV-GATA1 show

A

Infusions of rAAV-GATA1 increased the number of escape failures during the initial block of active avoidance testing, similar to the effects of inescapable stress exposure (Fig. 4d).

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40
Q

during the second block what did infusions of rAAV-GATA1 show and what does this mean

A

During the second block of active avoidance testing, there was no significant effect indicating that GATA1 delays responding, but does not produce a sustained effect in this model.

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41
Q

Infusion of rAAV-GATA1 had what effect on locomotor activity

A

Infusion of rAAV-GATA1 did not influence locomotor activity (not shown), indicating that there was no generalized effect on ambulation.

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42
Q

what did further studies show of the effects of rAAV-GATA1 in the forced swim test

A

Further studies showed that the effects of rAAV-GATA1 in the forced swim test were not reversed by the antidepressant imipramine as expected, since drug treatment would not influence viral expression of GATA1

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43
Q

How was the The influence of GATA1 on depressive behavior caused by chronic stress examined

A

 The influence of GATA1 on depressive behavior caused by chronic stress was examined with a viral knock down strategy, using a small hairpin RNA (shRNA) targeted to GATA1 (rAAV-GATA1shRNA) (Fig. 4e).

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44
Q

The ability of rAAV-GATA1shRNA to effectively decrease Gata1 mRNA was confirmed in what

A

in cultured cells and rat PFC

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45
Q

What does (rAAV-ScrshRNA) stand for

A

scrambled control

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46
Q

The rAAV-GATA1shRNA or scrambled control (rAAV-ScrshRNA) was infused into the PFC f rats that were then subjected to the CUS paradigm showed what

A

shown to increase Gata1 mRNA

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47
Q

The rAAV-GATA1shRNA or scrambled control (rAAV-ScrshRNA) was infused into the PFC f rats that were then subjected to the CUS paradigm showed what shown to increase Gata1 mRNA what did this paradigm result in

A

This paradigm results in anhedonia

48
Q

what is anhedonia and how can it be measured

A

a core symptom of depression that can be measured by preference for a sweetened solution

49
Q

CUS exposure significantly did what to rats and controls with rAAV-GATA1shRNA. infusion in sweetened solution and what was the effect in non-stressed rats. what did this show

A

 CUS exposure significantly decreased sucrose preference in control rats infused with rAAV-ScrshRNA and this effect was completely blocked by infusion of rAAV-GATA1shRNA.

 There was no effect of rAAVGATA1shRNA in non-stressed rats indicating that basal levels of GATA1 are low and not sufficient to suppress basal rates of sucrose preference

50
Q

At the presynaptic level, synapsin I, Rab3A and calmodulin 2 regulate what

A

regulate the the size, number, and targeting of synaptic vesicles,

51
Q

At the presynaptic level β-tubulins are invovled in what

A

axonal outgrowth and regeneration

52
Q

Postsynaptically, Rab4B regulates what

A

Postsynaptically, Rab4B regulates endosomal recycling that is required for spine maintenance and neurotransmitter receptor recycling

53
Q

what did the findings show

A

together these findings demonstrate molecular and cellular alterations that could underlie the reduction in neuronal cell body size and volume of PFC in MDD patients

54
Q

Decreased expression of these genes in response to chronic stress exposure suggests what

A

Decreased expression of these genes in response to chronic stress exposure also suggests an etiological relationship to MDD, which is often associated with severe life stress and trauma

55
Q

What is GATA1 and what is one of its roles

A
  • GATA1 was also identified as a transcriptional repressor that is increased in MDD and has binding elements in the promoter regions of the synapse related genes.
  • GATA1 is a member of a zinc finger family of transcription factors that are evolutionarily conserved and play important roles in embryonic development
56
Q

Where is gata-1 found and what is its role

A

Although originally characterized in hematopoietic and cardiac tissues, GATA transcription factors are also expressed in endocrine tissue and brain, and are reported to regulate neuronal differentiation during development

57
Q

overall finding in gata-1 levels in control and MDD

A

Although GATA1 levels are low in controls, expression is increased in MDD and in response to chronic stress.

58
Q

General finding of gata-1

A

Over expression of Gata1 was sufficient to cause dendrite atrophy and decreased synaptic protein expression in cultured cortical neurons.
• Moreover, expression of Gata1 in PFC was sufficient to produce depressive behaviors, while knock down of Gata1 completely blocked depressive, anhedonic behavior caused by CUS exposure.
• Together, these studies demonstrate that expression of Gata1 in the PFC is sufficient and necessary for the development of depressive behaviors in multiple animal paradigms.
• Further evidence for a role of GATA1 is provided by a recent report of a polymorphism in the GATA1 binding site of the promoter for interleukin 6, an inflammatory cytokine dysregulated in depression

59
Q

what do these results show in terms of anti-depressants

A
  • The results suggest that approaches that block or reverse neuronal atrophy in the PFC could be effective antidepressant treatments.
  • This possibility is supported by recent studies demonstrating that the rapid antidepressant actions of NMDA receptor blockade are associated with increased spine number and function, and increased synaptogenesis in the PFC
60
Q

How was Quantitative Real Time PCR conducted

A
  1. An aliquot of the total RNA that was previously extracted from prefrontal cortex punches was used for secondary validation using real time PCR.
  2. Five hundred ng of total RNA was used for cDNA synthesis using oligo dT primers and SuperScript II reverse transcriptase (Invitrogen), and subsequently diluted with nuclease-free water to 10 ng ul−1 cDNA.
  3. Genespecific high-melt temperature primers for genes of interest were designed using Primer 3 software and expressed sequence information obtained from GenBank (NCBI).
  4. PCR reactions were conducted on an ABI 7900 Sequence Detection System (Applied Biosystems) using a hotstart SYBR-green based method (Quantitect, QIAGEN) followed by melt curve analysis to verify specificity of product.
61
Q

what is the CT value

A

cycle number at threshold)

62
Q

what was the CT value used for

A

The CT value (cycle number at threshold) was used for calculations of relative amount of mRNA molecules.

63
Q

how was the CT value normalized and what is the value

A

The CT value of each target genes was normalized by subtraction of the CT value from multiple housekeeping genes.

 This value is the ΔCT.
64
Q

The difference in ΔCT between Control and MDD represents what and how was the relative quantitative change shown

A

The difference in ΔCT between Control and MDD represents the ΔΔCT, and the relative quantitative change was showed as 2−ΔΔCT

65
Q

All of the genes of interest were normalized to what gene

A

All of the genes of interest were normalized to the housekeeping gene, cyclophilin.

66
Q

in Electronic Microscopic Stereology how was the post-mortem samples prepared

A

Electronic Microscopic Stereology

  • The number of spine synapses in dlPFC of postmortem brains was counted as previously described
  • Briefly, postmortem samples were immersed overnight in a mixture of 2% glutaraldehyde + 4% paraformaldehyde dissolved in phosphate buffer.
67
Q

in Electronic Microscopic Stereology what occured with vibratome sections

A

Vibratome sections of 100 µm were cut throughout the tissue blocks and embedded in Durcupan

68
Q

in Electronic Microscopic Stereology what followed the vibratome sections

A

Using the embedded sections, at least ten sampling areas were randomly selected from each brain, and ultrathin sections (~70 nm) from each sampling area were prepared with a Reichert Ultracut E ultrotome. Digitized electron micrographs of each sampling area were taken in a Tecnai 12 transmission electron microscope

69
Q

in in Electronic Microscopic Stereology Digitized electron micrographs of each sampling area were taken in a what

A

Tecnai 12 transmission electron microscope

70
Q

what tequchine was used to count synapses in Electronic Microscopic Stereology

A

Synapses were counted using the dissector technique.

71
Q

in Electronic Microscopic Stereology synpase number for each brain was obtained how

A

Synapse numbers for each brain were obtained by averaging data from at least ten dissectors

72
Q

Sections of human brain and rat brain were sectioned / cut from what in in situ hybridization

A

in situ hybridization

• Sections of human brain and rat brain were cut from blocked dlPFC.

73
Q

following sectioning of the brain in in situ hybridization, what happened

A

brains stored at −80 °C.

74
Q

following brain stoarge in in situ hybridization, what happened

A

Brains were mounted onto ‘probe-on plus’ glass slides (Fisher Scientific)

75
Q

in situ hybridization how was the complimentary RNA probe (cRNA) prepared

A

To prepare complimentary RNA probe (cRNA) for in situ hybridization histochemistry, the fragments of cDNA were amplified by PCR using T7 promoter attached primer and PCR products were used to generate 35S-radiolabeled riboprobes using T7 RNA polymerase in vitro transcription (Ambion, T7-MEGAshortscript).

76
Q

in in situ hybridization PCR products were used to generate what and using what

A

PCR products were used to generate 35S-radiolabeled riboprobes using T7 RNA polymerase in vitro transcription (Ambion, T7-MEGAshortscript).

77
Q

in in situ hybridization, what happened after fixiation and acetylation

A

After fixation and acetylation, human or rat sections were hybridized with 35S-CTP incorporated RNA probes at 60 °C for 16 hr in the hybridization solution

78
Q

in in situ hybridization what happened to the sections affter fixiation and acetylation

A

Sections were then washed at 60 °C, dehydrated with graded alcohols, air-dried and exposed to film.

79
Q

Stress and antidepressant model

to look at For CUS, how was this measured in rats/controls

A

Stress and antidepressant model

  1. Rats were subjected to chronic unpredictable stress (CUS) for 35 days
  2. fluoxetine was administrated
  3. Control rats given for vehicle (saline) administration and brains were harvested for in situ hybridization analysis 4 hr after the last vehicle or drug treatment.
80
Q

in Stress and antidepressant model what happened to the brains after giving drug / saline

A

brains were harvested for in situ hybridization analysis 4 hr after the last vehicle or drug treatment.

81
Q

For stress study with rAAV-GATA1shRNArats how long were they subjected to CUS after viral infusion

A

3 weeks

82
Q

in Transcription factor binding motif search what was used to search binding motif

A

The scoring matrix of TRANSFAC was used for the binding motif search in promoter regions (1 kb of the 5’-flanking sequence and 200 bp on downstream from transcription start site) of our candidate genes.

83
Q

in Chromatin Immunoprecipitation Cultured primary cortical neurons (DIV 7) were incubated in what and for what

A

Chromatin Immunoprecipitation

• Cultured primary cortical neurons (DIV 7) were incubated in formaldehyde at RT for cross-linking of DNA and DNA binding proteins.

84
Q

in Chromatin Immunoprecipitation Crosslinked neurons were lysed in what

A

Crosslinked neurons were lysed in SDS lysis buffer ( SDS, EDTA, Tris-HCl, pH 8.1) including protease inhibitors and phosphatase inhibitors.

85
Q

in Chromatin Immunoprecipitation Chromatin was sheared by what

A

sonication

86
Q

in Chromatin Immunoprecipitation Chromatin was separated by what

A

centrifugation

87
Q

in Chromatin Immunoprecipitation what happened when the The supernatant was collected

A

The supernatant was collected and immunoprecipitated with dynabead (Dynal, 112-01D) conjugated with anti-GATA1 (Santa Cruz, sc-265) or normal horse serum overnight at 4 °C.

88
Q

in Chromatin Immunoprecipitation After reverse cross-linking, DNA was removed using what and quantified with what

A

After reverse cross-linking, DNA was removed using Qiagen minelute purification kit and quantified by real time PCR.

89
Q

in Primary cortical neuron culture what was Embryonic brains were prepared from and what happened after

A

Embryonic brains were prepared from fetal SD rat at 18 d gestation and mechanically triturated

90
Q

in Primary cortical neuron Dissociated cells were plated on 12 well plates in plating medium consisting of what

A

Dissociated cells were plated on 12 well plates in plating medium consisting of Neurobasal media supplemented with fetal bovine serum, L-glutamine, sodium pyruvate, HEPES, penstrep and B27 supplement.

91
Q

in Primary cortical neuron how was proliferation of non-neuronal cells stopped

A

Proliferation of non-neuronal cells was halted by replacing with plating medium lacking fetal serum in the next day (DIV 1).

92
Q

in Primary cortical neuron cultures were fed once a week with what

A

Cultures were then fed once a week with plating medium lacking fetal serum and maintained at 37 °C in a humidified 5% CO2 atmosphere.

93
Q

describe whole process of Primary cortical neuron

A

Embryonic brains were prepared from fetal SD rat at 18 d gestation and mechanically triturated
• Dissociated cells were plated on 12 well plates in plating medium consisting of Neurobasal media supplemented with fetal bovine serum, L-glutamine, sodium pyruvate, HEPES, penstrep and B27 supplement.
• Proliferation of non-neuronal cells was halted by replacing with plating medium lacking fetal serum in the next day (DIV 1).
• Cultures were then fed once a week with plating medium lacking fetal serum and maintained at 37 °C in a humidified 5% CO2 atmosphere.

94
Q

in Western blot what was Cultured neurons were lysed in

A

Western blot

  • Cultured neurons were lysed in RIPA buffer.
  • Protein levels were measured using BCA Protein Assay Kit.
  • Proteins were separated by SDS-PAGE and transferred to the nitrocellulose membranes.
  • After blocking with skim milk, the blots were incubated with primary antibodies overnight at 4 °C followed by horseradish peroxidase-conjugated secondary antibodies for 1 hr at RT.
  • Blots were visualized by enhanced chemiluminescence and exposed on films.
95
Q

in Western blot Protein levels were measured using what

A

BCA Protein Assay Kit.

96
Q

in Western blot protein was seperated with what and transfered where

A

Proteins were separated by SDS-PAGE and transferred to the nitrocellulose membranes.

97
Q

in Western blot Protein what happens when blockign with skim milk

A

After blocking with skim milk, the blots were incubated with primary antibodies overnight at 4 °C followed by horseradish peroxidas

98
Q

Blots were visualized by what

A

enhanced chemiluminescence and exposed on films.

99
Q

describe whole process of western blot

A

Cultured neurons were lysed in RIPA buffer.
• Protein levels were measured using BCA Protein Assay Kit.
• Proteins were separated by SDS-PAGE and transferred to the nitrocellulose membranes.
• After blocking with skim milk, the blots were incubated with primary antibodies overnight at 4 °C followed by horseradish peroxidase-conjugated secondary antibodies for 1 hr at RT.
• Blots were visualized by enhanced chemiluminescence and exposed on films.

100
Q

describe Immunohistochemistry process

A
  • Sections were fixed in paraformaldehyde-PBS and followed hydrogen peroxide treatment
  • Sections were incubated in a blocking solution , reacted with rabbit polyclonal anti-GFP or mouse monoclonal antibody recognizing MAP2 in PBS and then reacted with secondary antibody for 1 hr at RT.
101
Q

Dendritic branching was investigated by what

A

Sholl analysis

102
Q

Total dendritic length was estimated by what

A

Total dendritic length was estimated by counting the total number of circle intersections

103
Q

in Sholl analysis nsity of dendrites was defined by whhat

A

density of dendrites was defined by counting the number of intersections on each circles.

104
Q

in Sholl analysis the confocal images of neurons were analyzed using what

A

The confocal images of neurons were analyzed using Image J software which has plugged in Sholl analysis.

105
Q

describe process of Sholl analysis

A

Dendritic branching was investigated by Sholl analysis
• A transparent grid showing concentric circles was placed over the dendritic image, the smallest circle was centered in the soma and the distance between each circle was equivalent to 10 µm apart.
• Total dendritic length was estimated by counting the total number of circle intersections and the density of dendrites was defined by counting the number of intersections on each circles.
• The confocal images of neurons were analyzed using Image J software which has plugged in Sholl analysis.

106
Q

in Stereotaxic surgery and infusions Rats were anesthetized with what

A

xylazine and ketamine

107
Q

in Stereotaxic surgery and infusions Bilateral viral injections were performed with what

A

Bilateral viral injections were performed with coordinates relative to the Bregm

108
Q

in Stereotaxic surgery and infusions , what happened after Bilateral viral injections were performed

A

Needles were removed and the scalp incision was closed with wound clips.

109
Q

in Stereotaxic surgery and infusions what happened After the behavioral testing was performed,

A

animals were perfused with paraformaldehyde.

110
Q

in Stereotaxic surgery and infusions what happened after brain was perfused with paraformaldehyde

A

The brain was kept overnight in paraformaldehyde and then transferred to sucrose.

111
Q

in Stereotaxic surgery and infusions sections where cut using what and for what

A

sections were cut using a microtome for visualization of GFP.

112
Q

describe full process of Stereotaxic surgery and infusions s

A

Rats were anesthetized with xylazine and ketamin
Bilateral viral injections were performed with coordinates relative to the Bregm
• Needles were removed and the scalp incision was closed with wound clips.
• After the behavioral testing was performed, animals were perfused with paraformaldehyde.
• The brain was kept overnight in paraformaldehyde and then transferred to sucrose.
• sections were cut using a microtome for visualization of GFP.

113
Q

describe Learned Helplessness (LH) paradigm

A

Learned Helplessness (LH) paradigm

  • The LH procedure was performed in custom built, 2-chambered shuttle boxes (Med Associates, Vermont) .
  • For active avoidance testing, animals were exposed to 30 escape trials using an FR1 schedule in which a single crossing terminated the footshock.
  • Numbers of escape were automatically scored.
  • Results are expressed as number of escape failures observed.
114
Q

describe Forced swim test (FST)

A
  • On the test day, rats were placed in a clear cylinder with water .
  • The sessions were recorded from the side and the immobility time was scored by a blind observer.
  • Immobility was defined as floating or remaining motionless without leaning against the wall of the cylinder
115
Q

describe Locomotor Activity (LA)

A
  • To determine if there are general alterations in ambulation, locomotor activity was assessed.
  • Rats were placed in clear plastic chambers fitted with automated activity meters
  • Locomotor activity was recorded in bins
116
Q

describe Sucrose Preference test (SPT)

A
  • SPT was performed on day 21 after CUS.
  • Animals were habituated to sucrose solution followed by fluid deprivation on the test day.
  • Rats were presented with two identical bottles, one containing sucrose and the other water for 1 h and the consumption of each solution was measured.
  • Sucrose preference was calculated as the percentage of sucrose consumed over total fluid consumption.

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