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Brain Structure and Function

, Volume 224, Issue 1, pp 73–97 | Cite as

Infralimbic prefrontal cortex structural and functional connectivity with the limbic forebrain: a combined viral genetic and optogenetic analysis

  • Miranda Wood
  • Othman Adil
  • Tyler Wallace
  • Sarah Fourman
  • Steven P. Wilson
  • James P. Herman
  • Brent MyersEmail author
Original Article

Abstract

The medial prefrontal cortex is critical for contextual appraisal, executive function, and goal-directed behavior. Additionally, the infralimbic (IL) subregion of the prefrontal cortex has been implicated in stress responding, mood, and fear memory. However, the specific circuit mechanisms that mediate these effects are largely unknown. To date, IL output to the limbic forebrain has been examined largely qualitatively or within a restricted number of sites. To quantify IL presynaptic input to structures throughout the forebrain, we utilized a lentiviral construct expressing synaptophysin-mCherry. Thus, allowing quantification of IL efferents that are specifically synaptic, as opposed to fibers of passage. Additionally, this approach permitted the determination of IL innervation on a sub-structural level within the multiple heterogeneous limbic nuclei. To examine the functional output of the IL, optogenetic activation of IL projections was followed by quantification of neuronal activation throughout the limbic forebrain via fos-related antigen (Fra). Quantification of synaptophysin-mCherry indicated that the IL provides robust synaptic input to a number of regions within the thalamus, hypothalamus, amygdala, and bed nucleus of the stria terminalis, with limited input to the hippocampus and nucleus accumbens. Furthermore, there was high concordance between structural connectivity and functional activation. Interestingly, some regions receiving substantial synaptic input did not exhibit significant increases in Fra-immunoreactivity. Collectively, these studies represent a step toward a comprehensive and quantitative analysis of output circuits. This large-scale efferent quantification or ‘projectome’ also opens the door for data-driven analyses of the downstream synaptic mechanisms that mediate the integrative aspects of cortico–limbic interactions.

Keywords

Anterograde Fos-related antigen Rat Synaptophysin 

Abbreviations

3v

Third ventricle

A488

Alexa Fluor 488

aBST

Anterior BST

AAV

Adeno-associated virus

AHN

Anterior hypothalamic nucleus

AV

Anteroventral thalamus

BA25

Brodmann area 25

BLA

Basolateral amygdala

BMA

Basomedial amygdala

BST

Bed nucleus of the stria terminalis

BSTal

Anterolateral BST

BSTald

Dorsal portion of anterolateral BST

BSTalv

Ventral portion of anterolateral BST

BSTam

Anteromedial BST

BSTamd

Dorsal portion of anteromedial BST

BSTamv

Ventral portion of anteromedial BST

BSTfu

Fusiform BST

BSTif

Interfascicular BST

BSTov

Oval BST

BSTpr

Principal BST

BSTtr

Transverse BST

CA1

Cornu ammonis field 1

CA3

Cornu ammonis field 3

CaMKIIα

Ca2+/calmodulin-dependent protein kinase II alpha

cc

Corpus callosum

CeA

Central nucleus of the amygdala

CeL

Lateral subdivision of central amygdala

CeM

Medial subdivision of central amygdala

ChR2

Channelrhodopsin-2

CM

Centromedial thalamus

CoA

Cortical amygdala

DAPI

4′,6-Diamidino-2-phenylindole

DG

Dentate gyrus

DMH

Dorsomedial hypothalamus

fa

Corpus callosum anterior forceps

fx

Fornix

Fra

Fos-related antigen

GAD67

Glutamic acid decarboxylase, 67 kDa isoform

IHC

Immunohistochemistry

IL

Infralimbic cortex

LA

Lateral amygdala

LH

Lateral hypothalamus

LHab

Lateral habenula

mCh

mCherry

MD

Medialdorsal thalamus

MDD

Major depressive disorder

MeA

Medial amygdala

MeAad

Anterodorsal MeA

MeApd

Posterodorsal MeA

MeApv

Posteroventral MeA

MOC

Manders’ overlap coefficient

mPFC

Medial prefrontal cortex

MPN

Medial preoptic nucleus

mPOA

Medial preoptic area

mtt

Mammillothalamic tract

NAc

Nucleus accumbens

NAcC

NAc core

NAcS

NAc shell

NeuN

Neuronal nuclear protein

ot

Optic tract

pBST

Posterior BST

PH

Posterior hypothalamic nucleus

PL

Prelimbic cortex

PT

Paratenial thalamus

PVN

Paraventricular nucleus of the hypothalamus

PVT

Paraventricular thalamus

Re

Nucleus reunions

Sph

Synaptophysin

sm

Stria medullaris

st

Stria terminalis

VMH

Ventromedial hypothalamus

vSub

Ventral subiculum

YFP

Yellow fluorescent protein

Notes

Acknowledgements

AAV vectors were provided by the University of North Carolina Vector Core under material transfer agreement with Karl Deisseroth and Stanford University.

Funding

This work was supported by NIH grant K99/R00 HL122454 and an American Heart Association Fellowship to B. Myers, as well as NIH Grants R01 MH049698 and R01 MH101729 to J. P. Herman. T. Wallace received support from the Colorado State University Molecular, Cellular and Integrative Neuroscience program.

Compliance with ethical standards

Conflict of interest

The authors have no conflicts of interest to declare.

Ethical approval

All animal procedures and protocols were approved by the Institutional Animal Care and Use Committee and comply with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Miranda Wood
    • 1
  • Othman Adil
    • 1
  • Tyler Wallace
    • 2
  • Sarah Fourman
    • 1
  • Steven P. Wilson
    • 3
  • James P. Herman
    • 1
  • Brent Myers
    • 2
    Email author
  1. 1.Psychiatry and Behavioral NeuroscienceUniversity of CincinnatiCincinnatiUSA
  2. 2.Biomedical SciencesColorado State UniversityFort CollinsUSA
  3. 3.Pharmacology, Physiology, and NeuroscienceUniversity of South CarolinaColumbiaUSA

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