ABSTRACT
The basal forebrain (BF) is involved in arousal, attention, and reward processing but the role of individual BF subtypes is still being uncovered. Glutamatergic neurons are the least well-understood of the three major BF neurotransmitter phenotypes. Here we analyzed the distribution, size, calcium-binding protein content and projections of the major group of BF glutamate neurons expressing the vesicular glutamate transporter subtype 2 (vGluT2) and tested the functional effect of activating them. Mice expressing Cre recombinase under control of the vGluT2 promoter were crossed with a reporter strain expressing the red fluorescent protein, tdTomato, to generate vGluT2-cre-tdTomato mice. Immunohistochemical staining for choline acetyltransferase and a cross with mice expressing green fluorescent protein selectively in GABAergic neurons confirmed cholinergic, GABAergic and vGluT2+ neurons represent separate BF subpopulations. Subsets of BF vGluT2+ neurons expressed the calcium binding proteins calbindin or calretinin, suggesting that multiple subtypes of BF vGluT2+ neurons exist. Anterograde tracing using adeno-associated viral vectors expressing channelrhodopsin2- enhanced yellow fluorescent fusion proteins revealed major projections of BF vGluT2+ neurons to neighboring BF cholinergic and parvalbumin neurons, as well as to extra-BF areas involved in the control of arousal and regions responding to aversive or rewarding stimuli such as the lateral habenula and ventral tegmental area. Optogenetic activation of BF vGluT2 neurons in a place preference paradigm elicited a striking avoidance of the area where stimulation was given. Together with previous optogenetic findings suggesting an arousal-promoting role, our findings suggest BF vGluT2 neurons play a dual role in promoting wakefulness and avoidance behavior.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Funding: This work was supported by United States Veterans Administration Biomedical Laboratory Research and Development Service Merit Awards I01 BX004673, BX001356, I01 BX001404, I01 BX002774, I01 BX004500 and United States National Institute of Health support from NINDS R21 NS093000, NIMH R01 MH039683, NHLBI HL095491, R03-MH107650 and by SURE fellowships from Stonehill College. JTM, JMM, RB and REB are Research Health Scientists at VA Boston Healthcare System, West Roxbury, MA. The contents of this work do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.
Conflicts of interest: No conflicts of interest have been identified for any of the authors. JTM received partial salary compensation and funding from Merck MISP (Merck Investigator Sponsored Programs) but has no conflict of interest with this work.
Availability of data: Data is available upon written request
Table of abbreviations
- 3V
- Third ventricle
- A11
- A11 dopamine cells
- A12
- A12 dopamine cells
- A13
- A13 dopamine cells
- A14
- A14 dopamine cells
- AAV
- Adeno-associated virus
- aca
- Anterior commissure, anterior
- acp
- Anterior commissure, posterior
- ADP
- Anterodorsal preoptic nucleus
- AI
- Agranular insular cortex
- AMY
- Amygdala
- BF
- Basal forebrain
- BNST
- Bed nucleus of stria terminalis
- CA1
- Hippocampal field CA1
- Calb
- Calbindin Calr Calretinin
- CPu
- Caudate Putamen
- cc
- Corpus callosum
- Cg
- Cingulate cortex
- Cg1
- Cingulate cortex, area 1
- ChAT
- Choline acetyl transferase
- ChR2
- Channelrhodopsin 2
- Cl
- Claustrum
- cp
- Cerebral peduncle
- CTX
- Cotex
- DBv
- Vertical limb of the diagonal band
- DEn
- Endopiriform nucleus, dorsal
- DG
- Dentate gyrus
- DM
- Dorsomedial hypothalamic nucleus
- DP
- Dorsal peduncular cortex
- DR
- Dorsal raphe nucleus
- EYFP
- Enhanced yellow fluorescent protein
- f
- Fornix
- fmi
- forceps minor of the corpus callosum
- fr
- Fascilculus retrofelxus
- GAD67
- Glutamic acid decarboxylase-67
- GFP
- Green fluorescent protein
- GP
- Globus pallidus
- HDB
- horizontal limb of the diagonal band
- ic
- Internal capsule
- IEn
- Endipiriform cortex, intermediate
- IF
- Interfascicular nucleus
- IL
- Infralimbic cortex
- IMD
- Intermediodorsal thalamic nucleus
- LH
- Lateral hypothalamic area
- IPR
- Interpeduncular nulceus, rostral
- LDT
- Laterodorsal tegmental nucleus
- LHb
- Lateral habenula
- LM
- Lateral mammillary nucleus
- LO
- Lateral orbital cortex
- LP
- Lateral posterior thalamic nucleus
- LPO
- Lateral preoptic area
- LS
- Lateral septum
- LSUM
- Supramammillary nucleus, lateral
- M1
- Primary motor cortex
- M2
- Secondary motor cortex
- MCPO
- Magnocellular preoptic nucleus
- MD
- Mediodorsal thalamic nucleus
- Me
- Medial amygdaloid nucleus
- MHb
- Medial habenula
- ml
- medial lemniscus
- ML
- Medial mammillary nucleus, lateral
- MM
- Medial mammillary nucleus, medial
- MnPO
- Median preoptic nucleus
- MO
- Medial orbital cortex
- mp
- Mammillary peduncle
- MPA
- Medial preoptic area
- mPFC
- Medial prefrontal cortex
- MR
- Medial raphe nucleus
- MS
- Medial septum
- mt
- Mammillothalamic tract
- mtg
- Mammillotegmental tract
- NAc
- Nucleus accumbens
- ns
- Nigrostriatal bundle
- opt
- Optic tract
- PAG
- Periaqueductal gray
- PB
- Parabrachial nucleus
- PFC
- Prefrontal cortex
- Pir
- Piriform cortex
- Pm
- Principal mammillary tract
- PN
- Paranigral nucleus
- Po
- Posterior thalamic nuclear group
- PrL
- Prelimbic cortex
- PV
- Parvalbumin
- PVT
- Paraventricular thalamic nucleus
- RE
- Nucleus reuniens
- RLi
- Rotral linear raphe nucleus
- RPC
- Red nucleus, parvicellular
- SEM
- Standard error of the mean
- SH
- Septohippocampal nucleus
- SI
- Substantia innomminata
- SNC
- Substantia nigra, compacta
- SNR
- Substantia nigra, reticulate
- SUM
- Supramammillary nucleus
- TT
- Tenia tecta
- Tu
- Olfactory tubercle
- vGluT2
- Vesicular glutamate transporter 2
- VLPO
- Ventrolateral preoptic nucleus
- VM
- Ventromedial thalamic nucleus
- VMH
- Ventromedial hypothalamic nucleus
- VO
- Ventral orbital cortex
- VP
- Ventral pallidum
- VPT
- Ventral posterior thalamic nuclei
- VTA
- Ventral tegmental area of Tsai
- VTM
- Ventral tubermammillary nucleus
- ZI
- Zona incerta