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

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.

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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

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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.

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Correspondence to Brent Myers.

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The authors have no conflicts of interest to declare.

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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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Wood, M., Adil, O., Wallace, T. et al. Infralimbic prefrontal cortex structural and functional connectivity with the limbic forebrain: a combined viral genetic and optogenetic analysis. Brain Struct Funct 224, 73–97 (2019). https://doi.org/10.1007/s00429-018-1762-6

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Keywords

  • Anterograde
  • Fos-related antigen
  • Rat
  • Synaptophysin