Targeting Corticotropin-Releasing Factor Projections from the Oval Nucleus of the Bed Nucleus of the Stria Terminalis Using Cell-Type Specific Neuronal Tracing Studies in Mouse and Rat Brain

Abstract

The bed nucleus of the stria terminalis (BNST) is known to play a critical role in mediating the behavioural and autonomic responses to stressors. The oval nucleus of the BNST (BNSTov) contains cell bodies that synthesise the stress hormone corticotropin-releasing factor (CRF). Although afferent fibres originating from the BNSTov have been shown to innervate several key structures of the neuroendocrine and central autonomic system, the question remains as to whether some of these fibres are CRF-positive. To directly address this question, we injected a 'floxed' anterograde tracer (rAAV5/EF1a-DIO-mCherry) into the BNSTov of CRFp3.0Cre^GFP transgenic mice, which express a green fluorescent protein (GFP) under the control of the CRF promoter. Serial sections were then analysed for the presence of double-labelled fibres in potential projection sites. To determine whether CRF neurons in the rat BNSTov send comparable projections, we infused rat BNSTov with an adeno-associated viral vector (AAV) in which the human synapsin promoter drives enhanced GFP expression. We then used CRF immunoreactivity to examine double-labelled fluorescent fibres and axon terminals in projection sites from brain sections of the AAV-infused rats. We have observed several terminal fields in the mouse and rat brain with double-labelled fibres in the Dorsal raphe nucleus (DRD), the paraventricular nucleus of the hypothalamus and, to a lesser extent, in the ventral tegmental area. We found double-labelled terminal boutons in the nucleus accumbens shell, prelimbic cortex and posterior basolateral nucleus of the amygdala. The most intense double-labelling was found in midbrain, including substantia nigra pars compacta, red nucleus, periaqueductal grey and pontine nuclei, as well as DRD. The results of the present study indicate that CRF neurons are the output neurons of the BNSTov and they send projections not only to the centres of neuroendocrine and autonomic regulation, but also regions modulating reward and motivation, vigilance and motor function, as well as affective behaviour.

Keywords: bed nucleus of the stria terminalis; corticotropin-releasing factor; hypothalamus; oval nucleus; periaqueductal grey; raphe nucleus.

Figure 1

CRFp3.0Cre^GFP transgenic mice were injected with a floxed AAV5-DIO-mCherry viral vector into the BNSTov and analyzed for the presence of double-labeled BNSTov neurons. High level of co-expression between GFP- and m-Cherry-positive neurons was found in the BNSTov at the level of somata, dendrites, and axons (A–B″).

Figure 2

Double-labeled (GFP-mCherry) fibers originating from the BNSTov were found in the following projections sites in CRFp3.0Cre^GFP transgenic mice injected with a floxed AAV5-DIO-mCherry: dorsal raphé nucleus, dorsal division (DRD, A-A″, double arrows), the ventral tegmental area (VTA, B-B″, double arrows), and the paraventricular nucleus of the hypothalamus (PVN, C-C″, double arrows, scale bar 10 μm).

Figure 3

Rats were injected with the AAV5-hSyn-eGFP viral vector into the BNSTov (A) and neuronal tract tracing of the BNSTov neurons was combined with CRF-fluorescent immunoreactivity (CRF-ir, A″). Sub-population of eGFP-transfected BNSTov neurons co-expresses CRF at the level of somata, dendrites, and axons (A′-A‴). BNSTov neurons send abundant projections to the ventral (subcomissural) division of the BNST, including the fusiform nucleus (BNSTfus, A) but these fibers are not CRF-ir. Double-labeled fibers (eGFP-CRF, double-arrows) from the BNSTov were found entering posterior-medial BNST (BNSTpm,) through the stria terminalis (B-B″). Double-labeled fibers and perisomatic boutons formed around putative neurons were found in the nucleus accumbens shell (NAc, C-C″). In the PVN, double-labeled fibers were found in several PVN divisions including PaAP-anterior parvicellular part (D-D″, double arrows, scale bar 10 μm).

Figure 4

Intense eGFP-CRF double-labeled perisomatic baskets were found in the prelimbic cortex (PrL, A-A″, double arrows), whereas very limited eGFP-CRF co-expression was found in the cingulate cortex (CgC, B-B″). High level of eGFP labeling (C) and moderate CRF-ir (C′) was observed in the lateral hypothalamus (LH), and moderate eGFP-CRF co-localization was found in puncta and terminal boutons (C″). In contrast, although moderate e-GFP and CRF-ir was also observed in the paraventricular thalamus (PVT), very limited co-labeling was observed in the region (D″, scale bar 10 μm).

Figure 5

In the CeA (lateral part), high CRF-ir was found around putative neurons (A-A″, open arrows) and in the neuropil but limited CRF-ir boutons also co-expressed eGFP (A″, double arrows). In contrast, eGFP-CRF double-labeled puncta were found in the medial division of CeA (CeM, B-B″). High levels of CRF-ir were also found in the medial amygdala (MeA), but only sparse CRF-ir boutons also co-expressed eGFP (C-C″). In contrast, high level of CRF-eGFP co-expression was found in the posterior basolateral nucleus of the amygdala (BLA, D-D″, double arrows, scale bar 10 μm).

Figure 6

In the red nucleus, magnocellular part (RMC), CRF-eGFP double-labeled puncta made baskets around putative magnocellular neurons (A-A″, double arrows). However, some putative neurons are innervated by CRF independent of GFP (A″, open arrow). In the ventrolateral periaqueductal gray (VLPAG), double-labeled (CRF-eGFP) perisomatic contacts (puncta) were found around subsets of putative neurons (B-B″), indicative of potential CRF-containing axon-terminals from the BNSTov. In the brainstem, high-intensity and nearly complete co-localization of CRF-GFP positive fibers and boutons was found around putative neurons in pontine nuclei (PN, C-C″, double-arrows). In contrast, limited double-labeled boutons were found on puncta and boutons in the nucleus of the solitary track (SOL, double arrows, D-D″, scale bar 10 μm).

Figure 7

In the PVN (PaAP), eGFP-CRF-fibers (A-A″) were found in juxtaposition to local OT neurons (A-A‴prime;). Some of the OT neurons were also shown to co-express CRF (A′). In the VTA region, CRF-ir was found mostly independent of GFP in fibers and puncta. Sparse CRF-GFP terminals manifested as double-labeled puncta around cell bodies were found in the parabrachial pigmented nucleus (PBP) of the VTA, and the perisomatic contacts were found primarily with non-DA neurons, and only sporadic DA neurons (B-B‴′, TH-positive, double-arrows). In contrast, high level of CRF-GFP co-localization was found in the substantia nigra pars compacta (SNC) and double-labeled perisomatic contacts (puncta) were primarily formed around somata of DA-positive neurons (C-C‴, double-arrows). In the posterior substantia nigra reticulata (SNR), double-labeled contacts were found around both TH-negative and TH-positive, DA neurons (D-D‴, double arrows). In the dorsal raphé nucleus – dorsal division (DRD), double-immunofluorescent puncta (perisomatic baskets) were formed around a subset of 5-TPH-expressing, serotonergic neurons (E-E‴, double arrows, scale bar 10 μm).

Figure 8

Schematic representation of the CRF projections from the BNSTov.