Maternal deprivation induces a rapid decline in circulating leptin

levels and sexually dimorphic modifications in hypothalamic

trophic factors and cell turnoverViveros et. al.

Kristi Tschetter2/12/10

Overview of Chiaruttini et. al. • Coding region common to all BDNF transcipts that

contains a constitutively active dendritic targeting signal

• Targeting signal is suppressed in transcripts containing exons 1 or 4 these are resistricted to the soma and proximal dendrites

• Targeting signal is mediated by translin (RNA-binding protein implicated in RNA trafficking)

• Translin binding is disrupted by the G196A (or Val66Met mutation)

• G196A mutation blocks dendritic targeting of BDNF mRNA by disrupting its interaction with translin

BDNF Transcipts• Multiple BDNF transcripts are generated by

alternative splicing on one 5’ exon with a shared 3’ exon containing the entire BDNF coding region and either a long or a short 3’ UTR sequence

• Exon 1 and 4 transcripts are localized in the cell soma

• Exon 2 and 6 transcripts show a somatodendritic localization

• Splice variants appear to encode spatial localization signals used to preferentially regulate BDNF expression in different subcellular domains

Overall

• Translin mediates the constitutive dendritic targeting signal located in the CDS and this targeting mechanism is conserved in rat and human BDNF mRNA

• Evidence that translin/trax complex mediates dendritic targeting of BDNF mRNA

• BDNF coding region common to all BDNF slice variants is sufficient to direct constitutive targeting of BDNF mRNAs to the distal dendritic compartment

Figure 6

Figure 3

Figure 4

Results

• Characterization of this signal has demonstrated that1) Can be overridden by the 5’ UTRs of BDNF mRNA

isoforms (exon 1 and 4) that are retained in the cell body and proximal dendrites

2) Mediated by translin3) Blocked by the G196A (Val66Met) mutation

Results

• G196A substitution affinity impairs dendritic trafficking of BDNF mRNA through the disruption of its interaction with translin

• G196A SNP blocks dendritic trafficking of BDNF mRNA implies that phenotypic changes induced by this mutation, such as reduced hippocampal memory deficits complexity and volume, as well as memory deficits and susceptibility to mood disorders may be due to this effect

Discussion

• SNP may also impair BDNF protein sorting by disrupting its interaction with sortilin (a vasicular membrane protein implicated in membrane trafficking)

• G196A SNP interferes with BDNF processing by disrupting trafficking of both BDNF mRNA and protein through distinct mechanisms

Sirianni et. al. Overview

• BDNF is closed linked with neuronal survival and plasticity in psychiatric disorders

• Engineered a degradable, injectable alginate microspheres to continuously deliver BDNF to the dorsal hippocampus of rats for 2 days

• non-degradable implantable poly(ethylene vinyl acetate) matrices to continuously deliver BDNF to the dorsal hippocampus of rats for more than 1 week

Overview

• Small dose from a single infusion or from a 2 day sustained release alginate implant produced anti-depressant-like effects

• Prolonged delivery of BDNF resulted in a dysregulation of plasticity-related functions: increased dose and duration of BDNF delivery increased levels of TrkB, ERK, CREB, and phosphorylated ERK, while also producing decreased phosphorylated CREB

Overview

• Direct administration of neurotrophic factors in animals increases sprouting and growth of neurons

• Learning and adaptive deficiencies observed in depressive phenotypes are the consequence of decreased neuroplasticity due to loss of neurotrophic growth in the brain

• Loss of BDNF may underlie major depression• Antidepressant treatment is associated with

increased expression of BDNF

Goals

1) Examine behavioral and biochemical effects of BDNF delivery to the hippocampus

2) Develop drug delivery devices which could be used for future exploration of the biological effects of compounds that do not normally cross the blood brain barrier

Figure 1

Methods

• Stereotactic surgery– Target the CA1 and DG regions of the dorsal

hippocampus

Behavioral Test Methods

Open Field Forced Swim Test

Figure 2

Figure 3

Figure 4

Figure 5

Table 1

Table 2

Discussion

• EVAc and alginate materials were effective vehicles that could be used to deliver varying rates and doses of bioactive DNF to a local tissue region

• Advantages of alginate implants:– Allows for high loading of drug in polymer with

the use of an aqueous solvent– Doses can be administered by the same infusion

protocols used for fluid infusions

Discussion

• EVAc implant advantages– No drug is lost in the formulation steps and total

loading of drug in polymer is known exactly– Geometry of the implants can be altered in order

to target larger tissue regions– Can be designed to deliver sustained doses of

drug for longer periods of time than alginate materials

Results

• One-time bilateral infusion (25µg) of BDNF produced antidepressant-like effects in the forced swim test

• Delivery of BDNF from alginate microspheres was also antidepressant-like

• Delivery of BDNF from EVAc matrices was not antidepressant-like, even when the total dose was high (11µg)

Results

• BDNF function may be mediated via serotonergic action

• Selectively enhanced swimming behavior is consistent with other published reports demonstrating enhanced serotonergic function and the sprouting of survival of serotonergic neurons after the direct delivery of BDNF

• Sustained exposure to BDNF may interfere with its previously reported antidepressant-like effects

Results

• Two key features of BDNF– Relationship between neuroplasticity-related

behavior and neuroplasticity-related biochemistry is complex

– Precise effect of BDNF individual components of neuroplasticity-related processes depends on how it is delivered

• Dysregulation of BDNF-associated plasticity related pathways was observed after the sustained delivery of BDNF in the dorsal hippocampus of the rat

Overview of Viveros et. al.

• Circulating corticosterone levels were increased and glucose and leptin levels decreased throughout the study in both sexes

• Hypothalmic mRNA levels of leptin receptor increased significantly at MD24 in both sexes, normalizing in females at MD36, but not in males.

• Male rats insulin-like growth factor mRNA levels decreased at MD12 and MD24, with both trophic factors unaffected in females

Overview

• Males cell proliferation was significantly decreased at MD36, as were the glial structural proteins, glial fibrillary acidic protein and vimentin

• Females nestin levels decreased significantly at MD24

• MD differentially affects trophic factors and cell-turnover in the hypothalamus of males and females may underlie the sex differences seen in the endocrine and metabolic outcome

Background

• MS results in specific metabolic and hormonal alterations and delayed corporal growth

• MD ↑corticosterone levels exert changes on neurodevelopment including neurotrophic factors (NGF and BDNF), induces synaptic modifications, and ↑ neurodegeneration and possibly gliosis

Hypothesis

• During MD circulating and locally produced factors produced in the hypothalamic development are modified in a sexually dimorphic manner differentially affecting cell-turnover in the developing hypothalamus of males and females

Methods

• Adult Wistar rats• Reversed 12-h light-dark cycle (lights on at 20:00)• Free access to rat chow• On day of birth PND0, litters were culled to 8 pups per

dam (4 males and 4 females) did not cross foster• Total of 12 rats in each experimental group• Serum from all rats run in hormone analyses (n=11-12)• ½ hypothalami were used for protein analysis (n=6)

and ½ for mRNA analysis (n=6) with 2 rats from each of the 3 litters used in each analysis

Maternal Deprivation (MD)• On the morning of PND9 beginning at 9:00 mothers

from the deprived group were removed and placed in a cage beside the homecage in the same room

• For baseline measures and circadian variations pups of both sexes were sacrificed on the morning of PND9 at 9:00

• Both controls and test pups were killed 12h after the start of MD (Ct12 n=12 and MD12 n=12) and 24h after MD (Ct24 n=12 and MD24 n=24)

• On PND10, mothers were returned to the cage of their respective litters and 12h later another experimental group was sacrificed (Ct36, n=12 and MD36 n=16)

Figure 1A

Figure 1B

Figure 2A

Figure 2B

Figure 2C

Figure 3A

Figure 3B

Figure 3C and 3D

Figure 4

Figure 5A and 5B

Figure 5C and 5D

Figure 5E and 5F

Discussion

• Early MD has sexually dimorphic effects on the developing hypothalamus

• Basal levels of tropic factors such as IGF-1 and BDNF differ between the sexes and are modulated in a sex-specific manner during the separation period

• Sex differences in trophic factors are associated with differences in cell proliferation and cell specific markers

Discussion

• MD differentially affects cell development in the hypothalamus of male and female rats which could underlie the differences observed between the sexes in the pathological effects in the adult animal

• Both males and females have a significant increase in circulating corticosterone and a dramatic increase in leptin levels in response to MD which may be involved with some of the later endocrine outcomes

Detected Subcellular Localization Using in situ Hybridization

Localization of these transcipts parallels the transcripts in vivo even through they lack the BDNF 3’UTR

KCl did not effect the localization of any of the BDNF-GFP chimaeric

mRNAs or GFP-tubulin