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. Author manuscript; available in PMC: 2022 Nov 5.
Published before final editing as: Eur J Neurosci. 2021 May 5:10.1111/ejn.15260. doi: 10.1111/ejn.15260

Prefrontal Corticotropin-Releasing Factor Neurons Impair Sustained Attention via Distal Transmitter Release

Sofiya Hupalo 1,+, Robert C Spencer 1, Craig W Berridge 1,*
PMCID: PMC9215710  NIHMSID: NIHMS1814799  PMID: 33949025

Abstract

The prefrontal cortex (PFC) supports cognitive processes critical for goal-directed behavior. Although the PFC contains a high density of corticotropin-releasing factor (CRF) neurons, their role in cognition has been largely unexplored. We recently demonstrated that CRF neurons in the caudal dorsomedial PFC (dmPFC) of rats act to impair working memory via activation of local CRF receptors. However, there is heterogeneity in the neural mechanisms that support the diversity of PFC-dependent cognitive processes. Currently, the degree to which PFC CRF neurons impact other forms of PFC-dependent cognition is unknown. To address this issue, the current studies examined the effects of chemogenetic manipulations of PFC CRF neurons on sustained attention in male rats. Similar to working memory, activation of caudal dmPFC CRF neurons impaired, while inhibition of these neurons or global CRF receptor antagonism improved, sustained attention. However, unlike working memory, the sustained attention impairing effect of PFC CRF neurons was not dependent on local CRF receptors. Moreover, CRF infusion into the caudal dmPFC or other medial PFC subregions had no effect on task performance. Together, these observations demonstrate that while caudal dmPFC CRF neurons impair both working memory and sustained attention, these actions involve distinct neural circuits (local CRF release for working memory and extra-PFC release for sustained attention). Nonetheless, the procognitive actions of systemically administered CRF antagonists across both tasks are similar to those seen with attention deficit hyperactivity disorder-related treatments. Thus, CRF antagonists may have potential for use in the treatment of PFC cognitive dysfunction.

Keywords: corticotropin-releasing factor, prefrontal cortex, sustained attention, DREADDs

Graphical Abstract

graphic file with name nihms-1814799-f0001.jpg

Chemogenetic activation of caudal dmPFC CRF neurons impairs both sustained attention and working memory. Conversely, chemogenetic inhibition of these neurons or global administration of CRF antagonists improves these cognitive processes. However, the projection pathways involved in these actions are highly distinct, involving local CRF neurotransmission in the case of working memory and CRF release outside the PFC in the case of sustained attention.

Introduction

The prefrontal cortex (PFC) supports a diversity of cognitive processes that optimize goal attainment in a context-dependent manner (Fuster, 2015). PFC-dependent cognitive dysfunction is associated with multiple psychopathologies. However, the efficacy of current pharmacological treatments for these deficits is limited, likely reflecting our incomplete understanding of the neurobiology of PFC-dependent cognition (Millan et al., 2012). While it has long been known that corticotropin-releasing factor (CRF) receptors and neurons are prominent within the PFC (Swanson et al., 1983; De Souza et al., 1985; Uribe-Mariño et al., 2016), the role of PFC CRF in higher cognitive function has only recently begun to be examined. In prior studies, we demonstrated that CRF receptor activation in the rat caudal dorsomedial PFC (dmPFC) disrupts PFC-dependent working memory, while CRF receptor blockade, either locally in the caudal dmPFC or globally in the brain, improves working memory (Hupalo and Berridge, 2016). Subsequent chemogenetic studies determined that CRF neurons in the caudal dmPFC exert similar working memory effects that are dependent on local CRF receptors (Hupalo et al., 2019b).

PFC cognitive processes display heterogeneity in terms of sensitivity to neuromodulators (e.g. noradrenergic α1 receptors; Arnsten et al., 1999; Lapiz and Morilak, 2006; Alsene et al., 2011; Spencer and Berridge, 2019). Therefore, to fully understand the role of the PFC CRF system in cognition, it is important to examine how this system impacts distinct PFC-dependent cognitive processes. Bangasser and colleagues previously demonstrated that intracerebroventricularly (ICV) administered CRF elicits an impairment in sustained attention (Cole et al., 2016). However, it is unknown whether these actions reflect an involvement of CRF neurons and receptors within the PFC or if inhibition of CRF neurotransmission, globally and in the PFC, improves sustained attention. To address these issues, the current studies manipulated CRF neuronal and receptor activity within the PFC of animals tested in an operant task of sustained attention. This task requires discrimination of brief visual stimuli (‘signals’) from non-signal events (McGaughy and Sarter, 1995; Bushnell et al., 1997), is highly dependent on an intact PFC (Rueckert and Grafman, 1996; Pezze et al., 2014; Spencer and Berridge, 2019) and mirrors tests of sustained attention in human subjects (Bushnell, 1998; Sarter and McGaughy, 1998). To manipulate CRF neurotransmission within the PFC, we administered intra-PFC infusions of CRF and CRF antagonists and utilized a dual adeno-associated virus (AAV) chemogenetic approach to express excitatory (hM3Dq) or inhibitory (hM4Di) DREADDs selectively in PFC CRF neurons (Hupalo et al., 2019b). To better understand the potential translational relevance of CRF antagonists as procognitive treatments, we also examined the sustained attention effects of systemic and ICV administered CRF antagonists.

We observed that chemogenetic activation of CRF neurons in the caudal (but not rostral) dmPFC impairs sustained attention, while chemogenetic inhibition of this neuronal population improves sustained attention, mirroring that seen with working memory. However, in contrast to working memory, the sustained attention effects of these manipulations were not dependent on PFC CRF receptors. Moreover, direct activation of PFC CRF receptors had no impact on sustained attention performance. Thus, while CRF neurons in the caudal dmPFC impair both sustained attention and working memory, they do so through highly distinct pathways. The ability of systemic/ICV CRF antagonists to improve performance in both tests of working memory and sustained attention suggests CRF may represent a novel target for the treatment of PFC-dependent cognitive dysfunction.

Materials and Methods

Animals

Male Sprague-Dawley rats (300-500 grams; age 3-7 months; Charles River, Wilmington, MA) were pair-housed on a 13/11-hour light-dark cycle. Animals were fed 15-19 g chow/day to maintain motivation for food reward. Behavioral testing occurred 6 days/week. All facilities and procedures were in accordance with the National Institutes of Health and approved by the Institutional Animal Care and Use Committee at the University of Wisconsin-Madison.

Signal detection test of sustained attention

Animals were trained in an operant-based signal detection test of sustained attention (Berridge et al., 2006, 2012). All sessions consisted of 100 trials. On half of the trials (selected at random), a centrally located LED was illuminated at varying durations (“signal trials”). Following this, two levers projected into the chamber below the LEDs until a response was made and then retracted. On signal trials, a right lever press was scored as a “hit” and reinforced with one sucrose pellet (45 mg sucrose pellet/trial, Bio-Serv, Frenchtown, NJ), whereas a left lever press was scored as a “miss”. On the other half of trials (“no-signal trials”), the LED remained dark. On a no-signal trial, a left lever press was reinforced (“correct rejection”), while a right lever press was scored as a “false alarm.” The presence of interleaved signal and no-signal trials requires an animal to continuously monitor the stimulus location to guide behavior (e.g. press left or right lever). On correct trials, the house lights were illuminated for 5-sec with reward presentation. On incorrect trials, the levers retracted and a 5-sec time-out period ensued (houselights off). Trials were considered omitted if the animal failed to respond within 5-sec of lever projection. In this case, the levers were retracted followed by a 5-sec blackout. A variable intertrial interval (minimum 5-sec, 14-sec average) elapsed before the start of new trial. Animals were trained until proportion of correct trials reached 75%, which was followed by surgical implantation of cannulae and/or viral infusion.

Dependent measures included the probability of a hit (number of hits/number of signal trials), probability of a false alarm (number of false alarms/number of no-signal trials), response latency (sec between lever projection and lever press response), omissions (number of trials in which no response was made), and d’, a measure of stimulus detectability that takes into account both the probability of a hit and the probability of a false alarm (Gescheider, 1985; Berridge et al., 2012). d’ was calculated as follows: Z(N) - Z(SN); where Z(N) = 1 - probability of false alarms (z-score of the noise distribution), and Z(SN) = [(1-probability of a hit) + noise distribution] (z-score of the signal).

Surgery and viral infusions

Surgery.

All surgery was performed post-training and under isoflurane anesthesia (1-2%).

mPFC viral infusions.

For chemogenetic activation of PFC CRF neurons, a cocktail of two viruses was infused into either the rostral (A+4.4 to 3.4; L±0.8; V-2.2) or caudal (A+3.2 to +2.2; L±0.8; V-3.0) dmPFC. One virus drives CRF promoter-specific expression of Cre-recombinase (AAV8-CRF-Cre, 6.5X1013 gc/mL; Vector Biolabs, Malvern, PA), while the other drives Cre-dependent expression of hM3Dq or hM4Di (AAV8-hSyn-DIO-hM3Dq-mCherry, 4X1012 gc/mL; AAV8-hSyn-DIO-hM4Di-mCherry, 2.9X1013 gc/mL; Addgene, Cambridge, MA). A Cre-dependent virus lacking the DREADD transgene was used as a control (AAV8-hSyn-DIO-mCherry, 3X1012 gc/mL; Addgene, Cambridge, MA). The viral cocktail was mixed at a 1:2 ratio of CRF-Cre:DIO-mCherry, and 1.6 μL of this mixture was infused into the rostral or caudal dmPFC at a rate of 0.25 μL/min. After the infusion, 33-gauge stainless steel injector needles were kept in place for 3-min to allow for diffusion.

ICV, PFC or medial septal cannulae for drug infusions.

For all intra-brain infusions, 26-gauge stainless steel cannulae were surgically implanted 200 μm below the dura. ICV cannulae were implanted unilaterally and aimed at the lateral ventricle (A-0.8, L±1.5), with hemisphere counterbalanced. Some animals receiving viral infusions in the caudal dmPFC were also implanted with cannulae targeting either the caudal dmPFC (A +2.8; L ± 0.8) or the medial septum (MS; A+0.5, L±0.9, with 4° angle lateral from midline). Animals receiving rostral dmPFC viral infusions were also implanted with cannulae targeting this region (A +3.8; L ± 0.8). Four additional cohorts of animals received no viral infusions and were bilaterally cannulated for infusions targeting the rostral or caudal subfields of the dmPFC and ventromedial PFC (vmPFC). Regions were targeted using varying needle projection lengths (see below).

Drugs

CRF (human/rat, Bachem, Torrance, CA) was dissolved in buffered artificial extracellular fluid (Hupalo and Berridge, 2016). The non-selective CRF antagonist, D-Phe-CRF (human/rat, Bachem, Torrance, CA), the R1-selective CRF antagonist, NBI 35965, and the DREADD agonist, clozapine-N-oxide (CNO; NIMH Chemical Synthesis and Drug Supply Program), were dissolved in 0.9% saline. CNO was gently warmed immediately prior to injections to ensure solubility.

Treatments and testing

Animals first received a mock injection and/or intra-tissue infusion of vehicle 2 days prior to the first treatment to permit acclimation to infusion procedures and to minimize potential behavioral effects of tissue damage related to the initial needle insertion.

Treatments.

Rats were transported to the testing room in their home cage to receive a treatment and housed in their home cage for 15-min after the infusion. All intra-tissue infusions were performed with 33-gauge stainless steel needles. ICV infusions were made using needles projecting 2.0 mm past the cannula at a rate of 1 μL/min for 2-min (2 μL total). For the dmPFC, bilateral infusions were made using 2.0 mm (rostral dmPFC) or 3.0 mm (caudal dmPFC) projection lengths beyond cannulae. For the vmPFC, needles projected 4.0 mm. All intra-PFC infusions were made at a rate of 0.25 μL/min for 2-min (0.5 μL total). Bilateral intra-MS infusions were made using a projection length of 5.5 mm at a rate of 0.125 μL/min for 2-min (0.25 μL total). Needles were kept in place for 2-min following all infusions and then stylets were replaced. NBI 35965 (Million et al., 2003) and CNO were injected subcutaneously (1 mL/kg) 60-min and 45-min before testing, respectively. For dual systemic treatments, animals received a subcutaneous injection of NBI 35965 and 10-min later a second injection of CNO and tested 40-min later. All doses of a given drug treatment were counterbalanced. Every treatment was replicated and performance responses for the replicates were averaged.

Histology

Nissl Staining.

For experiments involving intracranial infusions, animals were deeply anesthetized and transcardially perfused with 3.7% wt/vol formaldehyde and brains stored in formaldehyde for at least 24-hrs before sectioning. Injector needle placement was verified in 40-μm thick coronal sections stained with Neutral Red dye (Sigma-Aldrich, St. Louis, MO).

Fluorescence microscopy.

For experiments involving chemogenetic manipulation of CRF neurons, brain sections were processed to visualize CRF-immunoreactivity (ir) and mCherry reporter protein fluorescence. Prior studies determined that this viral vector system permits selective manipulation of PFC CRF neurons (Hupalo et al., 2019b). As previously described, animals were perfused transcardially with chilled heparinized saline followed by 4% paraformaldehyde in 0.01 M phosphate buffer (Hupalo et al., 2019b). Brains were stored in paraformaldehyde overnight and dehydrated with graded 20-30% sucrose solutions. Immunohistochemical processing of CRF was performed using a primary guinea pig anti-CRF antibody (1:4000; catalog #t-5007; Peninsula Labs, San Carlos, CA) and a secondary donkey anti-guinea pig AF488 antibody (Jackson ImmunoResearch, West Grove, PA) as previously described (Hupalo et al., 2019b).

For animals receiving intra-PFC viral infusions and cannulae implants, alternating 40-μm thick sections were collected through the rostrocaudal extent of the PFC to confirm injector needle placement using Nissl staining and to examine viral spread using a BX51 Olympus light and reflected fluorescence microscope. Data from a given animal were included when histological analyses confirmed accurate placement of injector tracts, restriction of the transgene reporter protein (mCherry) to one PFC quadrant (rostral versus caudal dmPFC), and minimal tissue damage.

Statistical analyses

In general, the effects of a given treatment on sustained attention performance were compared to vehicle using a within-subject design. In studies examining the effects of ICV CRF and D-Phe-CRF, pilot studies were initially conducted to identify appropriate doses and/or confirm earlier observations (Cole et al., 2016). This resulted in the use of more than one cohort, with some animals not receiving every drug dose, while all doses were paired with a vehicle treatment. For these studies, data were analyzed with a linear mixed-effects model using the lmer package in R. For all other studies, treatment effects were compared to vehicle using a repeated-measures ANOVA. Additional analyses comparing the effects of CNO in hM3Dq animals to CNO-treated viral controls were done using a between subjects one-way ANOVA. Pairwise comparisons between drug dose and vehicle were calculated using Bonferroni corrected t-tests.

Results

Global CRF receptor actions in sustained attention

We first replicated earlier observations that ICV administration of CRF impairs sustained attention performance (Cole et al., 2016). We initially examined the effects of the highest dose of CRF (1 μg). When it was clear this affected performance, a second cohort of animals was used to expand the dose ranges, resulting in two cohorts, each receiving vehicle. Animals were treated ICV with vehicle (n=26), 0.025 μg (n=16), 0.2 μg (n=17), and 1 μg (n=8) CRF. One and two data points were eliminated from the 0.2 and 0.025 μg groups, respectively, due to infusion-related difficulties. Given not all subjects received all treatment doses in this experiment, drug effects were analyzed using a linear mixed effects model. As shown in Figure 1A and Table IA, ICV CRF elicited a dose-dependent impairment in sustained attention performance, reflected by decreases in d’ (F1,11.1 = 26.5; P < 0.001), proportion of hits (F1,49.1 = 8.4, P = 0.005) and increases in proportion of false alarms (F1,53.8 = 61.0, P = 0.001; Figure 1A, Table IA). Significant increases in response latency (F1,22.1 = 47.2, P = 0.001) and trial omissions (F1,21.8 = 11.4, P = 0.003) were also observed (Table IA). Interestingly, at the lowest dose tested (25 ng), ICV CRF elicited a small, but significant improvement in d’ scores (P = 0.02) without affecting other measures of performance (hits: P = 0.27, false alarms: P = 0.41; omissions: P = 0.69; response latency: P = 0.85).

Figure 1.

Figure 1.

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Effects of globally administered CRF and CRF antagonists on sustained attention. A) Effects of ICV administered vehicle (0 μg) or CRF (0.025 μg, 0.2 μg, 1 μg) on sustained attention performance as measured by d’. At the lowest dose, ICV CRF elicited a modest, yet statistically significant improvement in performance as measured by d’, while the 0.2 μg and 1 μg doses of CRF robustly impaired d’. B) Effects of ICV administration of the non-selective CRF1/2 antagonist, D-Phe-CRF (2 μg, 10 μg), on sustained attention D-Phe-CRF dose-dependently improved performance as measured by d’. C) Effects of systemically administered CRF1-selective antagonist, NBI 35965 (0.5 mg/kg, 2.5 mg/kg), on sustained attention. Bars represent mean ± SEM. *P < 0.05, **P < 0.01 vs. vehicle.

Table I.

Effects of ICV CRF and ICV/Systemic CRF Antagonists on Sustained Attention Performance.

A. ICV CRF
VEH 0.025 μg 0.2 μg 1.0 μg
Proportion of Hits 0.64 ± 0.03 0.66 ± 0.04 0.62 ± 0.03 0.50 ± 0.09**
Proportion of False Alarms 0.13 ± 0.01 0.1 ± 0.02 0.23 ± 0.03*** 0.37 ± 0.03***
Response Latency (s) 0.66 ± 0.04 0.60 ± 0.05 0.91 ± 0.06** 1.49 ± 0.22**
Trial Omissions 1.30 ± 0.69 2.47 ± 2.04 14.2 ± 4.71** 31.6± 8.76**
 
B. ICV CRF Antagonist (D-Phe-CRF)
VEH 2 μg 10 μg
Proportion of Hits 0.62 ± 0.03 0.63 ± 0.04 0.65 ± 0.03*
Proportion of False Alarms 0.12 ± 0.02 0.10 ± 0.02 0.11 ± 0.02
Response Latency (s) 0.60 ± 0.05 0.59 ± 0.06 0.54 ± 0.04*
Trial Omissions 1.24 ± 0.70 0.70 ± 0.30 0.39 ± 0.28*
 
C. Systemic CRF Antagonist (NBI 35965)
VEH 0.5 mg/kg 2.5 mg/kg
Proportion of Hits 0.68 ± 0.04 0.73 ± 0.05 0.75 ± 0.03
Proportion of False Alarms 0.08 ± 0.02 0.07 ± 0.02 0.05 ± 0.01
Response Latency (s) 0.72 ± 0.10 0.76 ± 0.08 0.67 ± 0.09
Trial Omissions 1.27 ± 0.78 1.65 ± 0.85 0.96± 0.49
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Values represent mean ± SEM for proportion of hits and false alarms, response latency and the number of trial omissions. A) Animals were treated ICV with vehicle (VEH) or CRF (0.025 μg, 0.2 μg, or 1.0 μg) or B) the non-selective CRF antagonist, D-Phe-CRF (2 μg, 10 μg). C) Animals were treated subcutaneously with VEH or the selective CRF1 antagonist, NBI 35965 (0.5 mg/kg, 2.5 mg/kg).

*

P < 0.05

**

P < 0.01

***

P < 0.001 vs. VEH (Bonferroni corrected t-test).

To assess whether blockade of CRF signaling globally in the brain impacts sustained attention, animals received ICV administered vehicle (n=18) or the non-selective CRF1/2 antagonist, D-Phe-CRF (2 μg, n=12; 10 μg, n=18). Doses of D-Phe-CRF were based on our previous studies (Hupalo and Berridge, 2016). As shown in Figure 1B, we observed a dose-dependent improvement in performance as measured by d’ that was significant at the 10 μg dose (F1,29.1 = 7.3, P = 0.01). This largely resulted from a significant increase in the proportion of hits (F1,29.0 = 4.6, P = 0.04; false alarms, F1,29.1 = 1.1, P = 0.3; Table IB). Additionally, ICV D-Phe-CRF dose-dependently decreased response latency (F1,29.1 = 6.2, P = 0.02) and trial omissions (F-1,26.2 = 4.3, P = 0.04; Table IB).

To better assess the potential translational utility of CRF antagonists, we also examined the sustained attention effects of systemic administration of the CRF1 antagonist, NBI 35965 (0.5, 2.5 mg/kg; n=13/group). Treatment with this antagonist significantly improved d’ in a dose-dependent manner (F2,12 = 4.5, P = 0.05; Figure 1C). This action was accompanied by non-significant increases in hits (F2,12 = 3.5, P = 0.09) and decreases in false alarms (F2,12 = 0.97, P = 0.34; Table IC). Response latencies (F2,12 = 3.6, P = 0.08) and trial omissions (F2,12 = 0.35, P = 0.56) were unaffected (Table IC).

Role of PFC CRF neurons in sustained attention

Additional studies examined whether dmPFC CRF neurons regulate sustained attention using a dual AAV chemogenetic approach (Figure 2). We focused on the dmPFC given our prior studies demonstrate that CRF manipulations in the vmPFC do not influence PFC-dependent cognition (Hupalo and Berridge, 2016). This pattern of cognitive actions is consistent with a well-documented dorsoventral functional topography within the medial PFC (Gabbott et al., 2005).

Figure 2.

Figure 2.

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Chemogenetic approach for bidirectional control of PFC CRF neurons. A) Schematic depicting dual viral system to activate PFC CRF neurons (CRF-Cre + hSyn-DIO-hM3Dq). Chemogenetic suppression of PFC CRF neurons is achieved using CRF-Cre + hSyn-DIO-hM4Di (not shown), while the control virus lacks the DREADD transgene (CRF-Cre + hSyn-DIO-mCherry; not shown). B) 40x photomicrograph of mCherry fluorescence in the caudal dmPFC from a CRF-hM3Dq-treated animal; scale bar = 250 μm; cc = corpus callosum. C) 400x photomicrographs demonstrating colocalization of mCherry fluorescence (red) with CRF-ir (green) in the caudal dmPFC; scale bar = 25 μm. All images are from a single section. The far-right panel is a merged image of both signals. 94 ± 5% of mCherry neurons colocalized with CRF-ir, and mCherry was not observed outside of CRF-ir neurons, as described previously (Hupalo et al., 2019b).

Activation of caudal dmPFC CRF neurons.

We first examined the effects of chemogenetic activation of caudal dmPFC CRF neurons (Figure 3). Four weeks after surgery, animals expressing hM3Dq in the caudal dmPFC received either vehicle or varying doses of systemic CNO (0.3, 3 mg/kg; n=8) and underwent testing. CNO dose-dependently impaired performance as measured by d’ relative to vehicle (F2,6 = 5.1, P = 0.02) and animals expressing viral controls lacking hM3Dq (n=6; F1,12 = 7.1, P = 0.02; Figure 3B). This was accompanied by a significant decrease in the proportion of hits (F2,6 = 3.7, P = 0.05, Table IIA). The proportion of false alarms (F2,6 = 1.8, P = 0.2), response latency (F2,6 = 2.4, P = 0.1), and trial omissions (F2,6 = 3.0, P = 0.1) were not significantly affected (Table IIA). To confirm the effects of CNO were dependent on CRF neurons in the caudal dmPFC, we examined the sustained attention effects of intra-PFC infusions of CNO. We observed similar effects with CNO directly infused into the caudal dmPFC (0.1, 1 mM; n=9), with a dose-dependent decrease in d’ (F2,7 = 7.3, P = 0.005; Figure 3B) and proportion of hits (F2,7 = 3.6, P = 0.05; Table IIB) relative to vehicle. As with systemic CNO, proportion of false alarms (F2,7 = 2.6, P = 0.1), response latency (F2,7 = 0.4, P = 0.7), and trial omissions (F2,7 = 0.43, P = 0.5) were unaffected (Table IIB).

Figure 3.

Figure 3.

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Sustained attention effects of chemogenetic activation and suppression of caudal dmPFC CRF neurons. A) Schematic depicting hM3Dq-CRF viral spread (dark shading) and CNO infusion sites (circles) within the caudal dmPFC of each animal (AP +3.2 to +2.2). B) Top, chemogenetic activation of caudal dmPFC CRF neurons using systemic CNO dose-dependently impairs d’ relative to vehicle (0 mg/kg, white bar) and viral controls (3 mg/kg, gray bar). Bottom, intra-PFC (iPFC) infusions of CNO similarly impaired d’ relative to vehicle (0 mM, white bar). C) Representative micrograph depicting the main body of a caudal dmPFC CNO infusion site. D) Chemogenetic suppression of caudal dmPFC CRF neurons (hM4Di) via systemic CNO injections dose-dependently improves d’ relative to vehicle (0 mg/kg, white bar). Bars represent mean ± SEM. **P < 0.01 vs. vehicle, ++P < 0.01 vs. viral controls.

Table II.

Effects of Chemogenetic Activation of CRF Neurons in the Caudal dmPFC on Sustained Attention Performance.

A. Systemic CNO + hM3Dq
VEH 0.3 mg/kg 3 mg/kg 3 mg/kg control
Proportion of Hits 0.69 ± 0.05 0.64 ± 0.04 0.57 ± 0.06 *+ 0.73 ± 0.04
Proportion of False Alarms 0.11 ± 0.03 0.1 ± 0.02 0.16 ± 0.06 0.07 ± 0.01
Response Latency (s) 0.65 ± 0.25 0.66 ± 0.25 0.93 ± 0.62 0.51 ± 0.13
Trial Omissions 1.00 ± 1.00 0.25 ± 0.16 5.21 ± 2.00 0 ± 0
 
B. iPFC CNO + hM3Dq
VEH 0.1 mM 1 mM
Proportion of Hits 0.65 ± 0.05 0.69 ± 0.04 0.6 ± 0.03*
Proportion of False Alarms 0.05 ± 0.02 0.1 ± 0.02 0.11 ± 0.02
Response Latency (s) 0.54 ± 0.19 0.55 ± 0.15 0.58 ± 0.23
Trial Omissions 1.33 ± 0.62 0.56 ± 0.56 1.28 ± 0.75
 
C. Systemic CNO + hM4Di
VEH 0.3 mg/kg 1 mg/kg
Proportion of Hits 0.59 ± 0.04 0.60 ± 0.04 0.63 ± 0.04
Proportion of False Alarms 0.11 ± 0.02 0.12 ± 0.03 0.09 ± 0.02
Response Latency (s) 0.64 ± 0.06 0.70 ± 0.07 0.65 ± 0.06
Trial Omissions 0.60 ± 0.3 0.50 ± 0.2 0.45 ± 0.13
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Values represent mean ± SEM for proportion of hits and false alarms, response latency and the number of trial omissions. A) Animals pretreated with the excitatory CRF-hM3Dq viral vector into the caudal dmPFC were tested after subcutaneous treatment with vehicle (VEH), 0.3 mg/kg, or 3.0 mg/kg CNO (first 3 columns). Data from animals pretreated with the control virus and given 3.0 mg/kg CNO are shown in the last column. B) Animals receiving the CRF-hM3Dq viral vector were tested following an intra-caudal dmPFC (iPFC) infusion of VEH, 0.1 mM, or 1 mM CNO. C) Animals pretreated with the inhibitory CRF-hM4Di viral vector into the caudal dmPFC were tested following subcutaneous administration of VEH, 0.3 mg/kg, or 3.0 mg/kg CNO.

*

P < 0.05 vs. VEH

+

P < 0.05 vs. viral control group receiving 3 mg/kg CNO.

Inhibition of caudal dmPFC CRF neurons.

To determine whether constitutive activity of caudal dmPFC CRF neurons influences sustained attention performance under normal testing conditions, we chemogenetically inhibited this neuronal population (Figure 3D). Animals infused with hM4Di virus into the caudal dmPFC were systemically treated with vehicle or CNO (0.3, 1 mg/kg; n=10). In these studies, CNO dose-dependently enhanced performance as measured by d’ (F2,18 = 4.3, P = 0.03; Figure 3D). Proportion of hits (F2,18 = 1.9, P = 0.32), false alarms (F2,18 = 1.8, P = 0.19), response latency (F2,18 = 2.5, P = 0.11), and trial omissions (F2,18 = 0.08, P = 0.9) were not significantly affected (Table IIC).

Activation of rostral dmPFC CRF neurons.

CRF neurons in the rostral dmPFC do not modulate working memory (Hupalo et al., 2019b). To assess whether this is true for sustained attention, we chemogenetically activated rostral dmPFC CRF neurons in a limited number of animals. Activation of rostral dmPFC CRF neurons (0.3, 3 mg/kg; n=5) with systemic CNO had no effects on task performance as measured by d’ relative to vehicle (F2,4 = 0.04, P = 0.8) and viral controls (n=4; F1,7 = 1.2, P = 0.3; Figure 4). Other measures of performance were also unaffected (hits, F2,4 = 2.7, P = 0.2; false alarms, F2,4 = 2.2, P = 0.2; response latency, F2,4 = 0.5, P = 0.6; trial omissions, F2,4 = 0.2, P = 0.7; Table IIIA). Intra-PFC infusions of CNO also had no significant effects on sustained attention performance (0.1, 1 mM; n=5; d’, F2,4 = 0.6, P = 0.5; hits, F2,4 = 0.2, P = 0.7; false alarms, F2,4 = 0.2, P = 0.6; response latency, F2,4 = 1.4, P = 0.3; trial omissions, F2,4 = 3.4, P = 0.1; Figure 4, Table IIIB).

Figure 4.

Figure 4.

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Chemogenetic activation of rostral dmPFC CRF neurons has no effect on sustained attention. Left, schematic depicting viral spread (dark shading) and CNO infusion sites (circles) in the rostral dmPFC for all animals (AP +3.4 to +4.4). Right, sustained attention effects (d’) of chemogenetic activation of rostral dmPFC CRF neurons via systemic (top panel) or intra-PFC (bottom panel) CNO treatment. Regardless of route of administration, CNO had no significant effects on d’ relative to vehicle (0 mg/kg, 0 mM, white bars) and viral controls (3 mg/kg, gray bar). Bars represent mean ± SEM.

Table III.

Effects of Chemogenetic Activation of CRF Neurons in the Rostral dmPFC on Sustained Attention Performance.

A. Systemic CNO + hM3Dq
VEH 0.3 mg/kg 3 mg/kg 3 mg/kg control
Proportion of Hits 0.50 ± 0.05 0.42 ± 0.08 0.58 ± 0.02 0.75 ± 0.05
Proportion of False Alarms 0.04 ± 0.02 0.04 ± 0.02 0.08 ± 0.04 0.06 ± 0.03
Response Latency (s) 0.29 ± 0.04 0.34 ± 0.07 0.32 ± 0.04 0.6 ± 0.16
Trial Omissions 0.20 ± 0.2 0.10 ± 0.1 0.20 ± 0.2 0 ± 0
 
B. iPFC CNO + hM3Dq
VEH 0.1 mM 1 mM
Proportion of Hits 0.49 ± 0.11 0.5 ± 0.11 0.47 ± 0.11
Proportion of False Alarms 0.06 ± 0.03 0.08 ± 0.04 0.07 ± 0.03
Response Latency (s) 0.35 ± 0.3 0.31 ± 0.03 0.36 ± 0.04
Trial Omissions 0.1 ± 0.1 0 ± 0 1.7 ± 0.94
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Values represent mean ± SEM for proportion of hits and false alarms, response latency and the number of trial omissions. A) Animals pretreated with the CRF-hM3Dq viral vector into the rostral dmPFC were tested following subcutaneous treatment with vehicle (VEH), 0.3 mg/kg, or 3.0 mg/kg CNO (first 3 columns). Data from animals pretreated with the control virus into the same region and then given 3.0 mg/kg CNO are shown in the last column. B) Animals pretreated with the CRF-hM3Dq viral vector into the rostral dmPFC were tested following an intra-caudal dmPFC (iPFC) infusion of VEH, 0.1 mM, or 1 mM CNO. There were no significant effects of CNO on any performance measures.

Potential involvement of CRF neurons in the MS.

For intra-caudal dmPFC viral infusions, mCherry expression was largely confined to this region. As noted previously (Hupalo et al., 2019b), in ~30% of animals we observed retrograde mCherry labeling of cell bodies in the MS (Figure 5A), an area that projects to the PFC (Senut et al., 1989). To assess whether CRF neurons in the MS contribute to the sustained attention effects of systemic CNO, 250 nl infusions of either CNO (1 mM) or vehicle were made into the MS prior to testing in a limited number of animals. In animals subsequently confirmed to display mCherry expression within the MS (n=6), intra-MS CNO had no effect on sustained attention performance as measured by d’ (F1,5 = 0.44, P = 0.5; Figure 5B-C) or any other measure relative to vehicle (data not shown).

Figure 5.

Figure 5.

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Chemogenetic activation of retrogradely-labeled CRF neurons in the MS has no effect on sustained attention. A) Retrograde mCherry cell body labeling was observed in the MS in ~30% of animals; scale bar = 200 μm. B) Schematic depicting the main body of CNO infusion sites (circles) into the MS of animals that were subsequently determined to have mCherry expression in this region. C) Intra-MS (iMS) CNO infusions in these animals had no significant effects on d’ relative to vehicle (0 mM). Bars represent mean ± SEM.

Role of CRF receptors in the sustained attention modulating actions of caudal dmPFC CRF neurons

To verify that the sustained attention impairing actions of caudal dmPFC CRF neuronal activation are CRF receptor-dependent, we pre-treated a subset of animals with systemic vehicle or the CRF1 selective antagonist, NBI 35965, prior to CNO-induced activation of caudal dmPFC CRF neurons (via 3 mg/kg CNO; n=9). In animals treated with systemic vehicle, CNO significantly impaired performance as measured by d’ (P = 0.006; Figure 6A). At a dose that on its own had no effect on task performance (1 mg/kg, P = 0.8), NBI 35965 completely prevented the sustained attention-impairing effects of CNO (CNO x Antagonist interaction, F1,8 = 7.9, P = 0.02).

Figure 6.

Figure 6.

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PFC CRF neurons do not impair sustained attention via local CRF receptors. A) In vehicle (VEH) pretreated animals (left bars), chemogenetic activation of caudal dmPFC CRF neurons (via 3 mg/kg CNO) significantly impaired sustained attention as measured by d’. In contrast, systemic pretreatment with the CRF1 antagonist, NBI 35965 (1 mg/kg; right bars) prevented this effect. B) In animals pretreated with vehicle (VEH) directly into the PFC (left bars), chemogenetic activation of caudal dmPFC CRF neurons (via 3 mg/kg CNO) significantly impaired sustained attention as measured by d’. However, infusion of the CRF antagonist, D-Phe-CRF (100 ng/hemisphere; right bars) directly into the caudal dmPFC prior to CNO failed to reverse this effect. Bars represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle.

To determine whether the sustained attention impairing actions of caudal dmPFC CRF neurons involve local CRF release and subsequent activation of local receptors, animals received intra-caudal dmPFC infusions of vehicle or the CRF1/2 antagonist, D-Phe-CRF (n=9), prior to systemic CNO administration (via 3 mg/kg CNO). In animals treated with intra-PFC vehicle, CNO impaired task performance as measured by d’ (P = 0.001; Figure 6B). Intra-PFC infusion of D-Phe-CRF at a dose that had no significant effect on d’ on its own (100 ng/hemisphere, P = 0.9) elicited no significant effect on CNO-induced impairment in sustained attention (CNO x Antagonist interaction, F1,8 = 2.7, P = 0.1).

These observations indicate CRF receptors in the caudal dmPFC may not impact sustained attention. To fully assess this, we examined the sustained attention effects of bilateral intra-caudal dmPFC infusions of vehicle or varying doses of CRF (25, 50, 250 ng; n=10). At doses that significantly impaired working memory (Hupalo and Berridge, 2016), CRF infusions into the caudal dmPFC had no impact on sustained attention as measured by d’ (F3,9 = 0.08, P = 0.8; Figure 7A) or any other performance measure (data not shown). Lastly, to assess whether CRF neurons within the caudal dmPFC might influence sustained attention via projections to other medial PFC subfields, we examined the sustained attention effects of CRF infused into the rostral dmPFC as well as the rostral and caudal ventromedial PFC (vmPFC). As shown in Figure 7B, CRF infusions into the rostral dmPFC failed to affect sustained attention performance as measured by d’ relative to vehicle (n=13; F3,12 = 0.01, P = 0.9). Similarly, when infused into the rostral (n=8/group) or caudal (n=5/group) vmPFC, CRF had no noticeable impact on sustained attention performance. When data from both vmPFC subregions were grouped, intra-vmPFC infusions of varying doses of CRF (n=13) had no significant effects on d’ (F3,11 = 1.2, P = 0.3; Figure 7C) or any other performance measure as compared to vehicle (data not shown).

Figure 7.

Figure 7.

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CRF receptor activation in varying PFC subfields has no effect on sustained attention. Shown are the sustained attention effects, as measured by d’, of CRF infusions into the A) caudal dmPFC, B) rostral dmPFC, and C) rostral + caudal subfields of the vmPFC. CRF infusions into these subregions of the medial PFC failed to impact sustained attention relative to vehicle (0 ng/hemisphere). Bars represent mean ± SEM.

Discussion

These studies demonstrate that CRF neurons in the caudal, but not rostral, dmPFC impair sustained attention, similar to that seen with working memory (Hupalo et al., 2019b). In contrast to that seen with working memory, the sustained attention impairing effect of caudal dmPFC CRF neuronal activation was not dependent on local CRF receptors and direct activation of PFC CRF receptors had no impact on sustained attention performance. We conclude that while caudal dmPFC CRF neurons exert similar impairing effects across different PFC-dependent cognitive processes, these actions involve distinct projection pathways. The existence of divergent circuits to regulate multiple PFC cognitive processes may allow for a more robust regulation of goal-directed behavior in a context-specific manner.

These studies also demonstrate that inhibition of CRF neurotransmission globally in the brain, via ICV or systemic administration of CRF antagonists, improves sustained attention performance similar to that seen with working memory (Hupalo and Berridge, 2016; Hupalo et al., 2019b). The procognitive actions of CRF antagonists are similar to those seen with attention deficit hyperactivity disorder (ADHD) medications (Berridge et al., 2012), suggesting that CRF antagonists may represent a novel pharmacological approach in the treatment of ADHD and/or other PFC-dependent cognitive disorders (Hupalo et al., 2019a). Collectively, these observations provide translationally relevant and novel insight into the neurobiology of PFC-dependent cognition.

Translational relevance: CRF antagonists as cognitive enhancers

Cognitive dysfunction is a core symptom in a variety of psychiatric disorders, including ADHD, depression, and schizophrenia. Yet, our ability to treat cognitive deficits associated with these disorders is limited (Millan et al., 2012). In the case of ADHD, all approved treatments have been shown to improve a diversity of PFC-dependent processes in ADHD patients as well as healthy human and animal subjects (Spencer et al., 2015). Thus, the observation that CRF antagonists improve performance in two distinct tasks of PFC-dependent cognition suggests these compounds might have clinical utility in the treatment of PFC cognitive dysfunction associated with ADHD. Given previous efforts to develop CRF1 antagonists for anxiety and major depressive disorder, many of these compounds have already passed safety tests in healthy human subjects (Kehne and Cain, 2010; Koob and Zorrilla, 2012). Clinical trials concluded that CRF antagonists are not effective in treating anxiety and depression, despite positive results observed in preclinical tests (Binneman et al., 2008; Coric et al., 2010; Kwako et al., 2015; Schwandt et al., 2016). This has highlighted the potential limitations of tests of anxiety and models of affective dysfunction in animals (Spierling and Zorrilla, 2017). In contrast, the working memory and sustained attention tests used to demonstrate the procognitive actions of CRF antagonists in animals align well with cognitive tests used in human subjects and accurately predict the cognitive effects of environmental (stress) and pharmacological manipulations seen in human subjects (Bushnell et al., 2003; Berridge et al., 2012; Arnsten, 2015). Indeed, the approval of the noradrenergic α2 agonist, guanfacine, for the treatment of ADHD was in part based on its working memory-enhancing actions observed in rodents and nonhuman primates (Tanila et al., 1996; Franowicz and Arnsten, 1998; Gamo et al., 2010). Thus, the cognitive tasks used with CRF antagonists appear to have strong translational relevance. Additional studies are needed to better understand the potential clinical utility of CRF antagonists in the treatment of PFC-dependent cognitive dysfunction in humans.

CRF1 is the predominant receptor subtype in the rodent PFC, although limited evidence suggests PFC CRF2 receptors may also be functionally relevant (De Souza et al., 1985; Van Pett et al., 2000; Robinson et al., 2019; Yarur et al., 2020). In the present study, both ICV CRF1/2 and systemic CRF1 antagonists improved sustained attention performance as measured by d’. However, while ICV CRF1/2 blockade additionally decreased response latency and trial omissions, CRF1 blockade did not. This suggests that both CRF receptor subtypes may modulate different aspects of sustained attention performance. Given the sustained attention impairing effects of caudal dmPFC neurons were blocked by CRF1 antagonism, it’s possible CRF may modulate sustained attention via additional circuitry involving CRF2. The precise role of CRF2 receptors in PFC-dependent cognition is unknown and further research is needed to determine whether selective CRF2 blockade elicits cognition-enhancing effects.

Lastly, stress and stress-related psychiatric disorders are associated with an impairment in PFC-dependent cognition (Arnsten, 2009). Recent observations suggest that PFC CRF systems are impacted in stress and may play a role in stress-related cognitive impairment (Meng et al., 2011; Uribe-Mariño et al., 2016). In the current studies, chemogenetic inhibition of caudal dmPFC CRF neurons improved sustained attention, indicating these neurons are constitutively active during testing. However, this is unlikely to reflect stress-related activation of these neurons given all animals were highly habituated to the testing procedures and readily participated in the task to obtain food rewards while displaying high baseline accuracy. Collectively, these results suggest that caudal dmPFC CRF neurons are active during conditions associated with high motivation and arousal, including, but not limited to, stress.

Sex differences in CRF modulation of sustained attention

The current study utilized male animals in part to align with a large body of work describing the neurobiology underlying sustained attention in males (McGaughy et al., 1996; Aston-Jones et al., 1999; Sarter et al., 2001; Spencer and Berridge, 2019). Available evidence indicates that PFC-dependent executive function, including sustained attention, displays minimal sex-dependent differences (McGaughy and Sarter, 1999; Grissom and Reyes, 2019). However, evidence indicates that females display differential sensitivity to certain behavioral and cellular actions of CRF in subcortical regions (Valera et al., 2010; Valentino et al., 2012; Howerton et al., 2014; Hupalo et al., 2019a). Additionally, limited evidence indicates females may be somewhat more sensitive to the sustained attention impairing effects of ICV CRF and ovarian hormones may protect against this action (Cole et al., 2016; Bangasser et al., 2019). Thus, it is currently difficult to speculate whether there exist sex differences in the cognitive actions of PFC CRF neurons and receptors. Ongoing studies in our laboratory are examining this issue.

Functional topography of PFC-dependent cognitive processes

These results add to a growing body of literature that documents heterogeneity in the circuit and receptor mechanisms which regulate PFC-dependent cognitive processes. The observation that CRF neurons in the dmPFC modulate working memory and sustained attention is consistent with a well-known dorsoventral topography within the PFC. Specifically, dorsal PFC subregions help support higher cognitive functions while ventral regions are more closely associated with emotional and motivational processes (Voorn et al., 2004; Hoover and Vertes, 2007). Additionally, our studies highlight the functional topography of the PFC in the rostrocaudal dimension (Bedwell et al., 2017). The observation that caudal, but not rostral, dmPFC CRF neurons impair working memory and sustained attention is similar to noradrenergic modulation of sensorimotor gating in rodents, in which α1 receptor activation in the caudal, but not rostral, dmPFC disrupts prepulse inhibition (Alsene et al., 2011). Moreover, converging anatomical evidence from rodents and non-human primates demonstrates that PFC projections to the striatum and thalamus are organized rostrocaudally (Sesack et al., 1989; Haber, 2003; Mailly et al., 2013; Vogelsang and D’Esposito, 2018). Lastly, evidence supports the existence of functional and anatomical organization along the rostrocaudal axis of the frontal cortex in human and non-human primates. For example, rostral prefrontal subfields are functionally activated during more abstract, higher-order processing, while activation of caudal/premotor subfields is associated with motor representations and actions (Dixon et al., 2014; Thiebaut de Schotten et al., 2017; Amiez and Petrides, 2018; Borra et al., 2019). As such, it has been posited that the rostrocaudal prefrontal axis in primates represents a hierarchical organization of goal-directed behavior (Badre and D’Esposito, 2009). The extent to which these observations align with the rodent PFC is currently unknown.

Neurocircuitry underlying the sustained attention actions of caudal dmPFC CRF neurons

The present studies suggest that at least a subset of PFC CRF neurons are projection neurons. Consistent with this, recent observations indicate that CRF is present in both inhibitory and excitatory neurons in the PFC (Kubota, 2014; Itoga et al., 2019). Moreover, in recently completed immunohistochemical studies, we observed that ~85% of PFC CRF neurons are immunoreactive for CaMKIIα (glutamatergic/pyramidal neuron marker), while 15% are immunoreactive for GAD67 (GABAergic/interneuron marker; unpublished observations). Available evidence indicates CRF neurons are present throughout the rostrocaudal and dorsoventral axes of the rat PFC (Swanson et al., 1983; Merchenthaler, 1984; Schreiber et al., 2017; Itoga et al., 2019). However, the distribution of excitatory vs. inhibitory CRF neurons across the rostrocaudal axis of the dmPFC remains to be definitively characterized.

Beyond functional topography PFC CRF neurons, our observations demonstrate that caudal dmPFC CRF receptors impair working memory performance but not sustained attention. This does not reflect differences in the degree to which these tasks are dependent on the dmPFC, as both tasks are highly, and comparably, dependent on this region (Spencer et al., 2012; Spencer and Berridge, 2019). The differential effects of CRF receptors across cognitive tasks are similar to that observed with PFC noradrenergic α1 reptors, which improve focused (sustained) and flexible attention (Lapiz and Morilak, 2006; Berridge et al., 2012), while impairing working memory (Arnsten et al., 1999). Collectively, these observations provide strong evidence for receptor-specific actions across distinct PFC-dependent cognitive processes.

There are multiple regions that possess CRF receptors and could play a role in the attention modulating actions of PFC CRF neurons, including cortical, thalamic, basal forebrain, and brainstem areas (Aston-Jones et al., 1999; Van Pett et al., 2000; Sarter et al., 2001; Sauvage and Steckler, 2001; Gritton et al., 2016; Schmitt et al., 2017). In the present studies, low visibility of mCherry-labeled axonal fibers precluded us from unambiguously identifying the output targets of caudal dmPFC neurons, potentially due to low anterograde transport of the AAV8 serotype. Nonetheless, the current observations provide a strong rationale for future studies to investigate the pathways involved in the sustained attention actions of PFC CRF neurons. Toward this goal, we have initiated studies combining AAV8-CRF-Cre with AAV5-DIO-eGFP, which has allowed us to better identify the anatomical projection targets of caudal dmPFC neurons. Using this approach, preliminary observations indicate the presence of dense terminal fields within the dorsomedial thalamus and medial parietal cortex, two regions implicated in attentional processes, following infusion of this viral cocktail into the caudal dmPFC. Ongoing studies are beginning to characterize these projection pathways and to determine the involvement of these terminal fields in the sustained attention impairing actions of caudal dmPFC CRF neurons.

Acknowledgements

We would like to thank Andrea Martin, Christopher Johns, Kayla Burton, and Yanya Ding for their dedicated technical assistance in this work.

Funding and Disclosure

This work was supported by the National Institutes of Health (MH081843 and MH102211 to C.W.B; MH107140 to S.H.); the Office of the Vice Chancellor for Research and Graduate Education at the University of Wisconsin-Madison; and funding from the Wisconsin Alumni Research Foundation.

Footnotes

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Alsene KM, Rajbhandari AK, Ramaker MJ, Bakshi VP (2011) Discrete forebrain neuronal networks supporting noradrenergic regulation of sensorimotor gating. Neuropsychopharmacology 36:1003–1014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amiez C, Petrides M (2018) Functional rostro-caudal gradient in the human posterior lateral frontal cortex. Brain Struct Funct 223:1487–1499. [DOI] [PubMed] [Google Scholar]
  3. Arnsten AFT (2009) Stress signalling pathways that impair prefrontal cortex structure and function. Nat Rev Neurosci 10:410–422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arnsten AFT (2015) Stress weakens prefrontal networks: molecular insults to higher cognition. Nat Neurosci 18:1376–1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Arnsten AFT, Mathew R, Ubriani R, Taylor JR, Li B-M (1999) α-1 noradrenergic receptor stimulation impairs prefrontal cortical cognitive function. Biol Psychiatry 45:26–31. [DOI] [PubMed] [Google Scholar]
  6. Aston-Jones G, Rajkowski J, Cohen J (1999) Role of locus coeruleus in attention and behavioral flexibility. Biol Psychiatry 46:1309–1320. [DOI] [PubMed] [Google Scholar]
  7. Badre D, D’Esposito M (2009) Is the rostro-caudal axis of the frontal lobe hierarchical? Nat Rev Neurosci 10:659–669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bangasser DA, Eck SR, Ordoñes Sanchez E (2019) Sex differences in stress reactivity in arousal and attention systems. Neuropsychopharmacology 44:129–139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bedwell SA, Billett EE, Crofts JJ, Tinsley CJ (2017) Differences in anatomical connections across distinct areas in the rodent prefrontal cortex Bolam P, ed. Eur J Neurosci 45:859–873. [DOI] [PubMed] [Google Scholar]
  10. Berridge CW, Devilbiss DM, Andrzejewski ME, Arnsten AFT, Kelley AE, Schmeichel B, Hamilton C, Spencer RC (2006) Methylphenidate Preferentially Increases Catecholamine Neurotransmission within the Prefrontal Cortex at Low Doses that Enhance Cognitive Function. Biol Psychiatry 60:1111–1120. [DOI] [PubMed] [Google Scholar]
  11. Berridge CW, Shumsky JS, Andrzejewski ME, McGaughy JA, Spencer RC, Devilbiss DM, Waterhouse BD (2012) Differential Sensitivity to Psychostimulants Across Prefrontal Cognitive Tasks: Differential Involvement of Noradrenergic α1- and α2-Receptors. Biol Psychiatry 71:467–473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Binneman B, Feltner D, Kolluri S, Shi Y, Qiu R, Stiger T (2008) A 6-Week Randomized, Placebo-Controlled Trial of CP-316,311 (a Selective CRH 1 Antagonist) in the Treatment of Major Depression. Am J Psychiatry 165:617–620. [DOI] [PubMed] [Google Scholar]
  13. Borra E, Ferroni CG, Gerbella M, Giorgetti V, Mangiaracina C, Rozzi S, Luppino G (2019) Rostro-caudal Connectional Heterogeneity of the Dorsal Part of the Macaque Prefrontal Area 46. Cereb Cortex 29:485–504. [DOI] [PubMed] [Google Scholar]
  14. Bushnell PJ (1998) Behavioral approaches to the assessment of attention in animals. Psychopharmacology (Berl) 138:231–259. [DOI] [PubMed] [Google Scholar]
  15. Bushnell PJ, Benignus VA, Case MW (2003) Signal detection behavior in humans and rats: a comparison with matched tasks. Behav Processes 64:121–129. [DOI] [PubMed] [Google Scholar]
  16. Bushnell PJ, Oshiro WM, Padnos BK (1997) Detection of visual signals by rats: effects of chlordiazepoxide and cholinergic and adrenergic drugs on sustained attention. Psychopharmacology (Berl) 134:230–241. [DOI] [PubMed] [Google Scholar]
  17. Cole RD, Kawasumi Y, Parikh V, Bangasser DA (2016) Corticotropin releasing factor impairs sustained attention in male and female rats. Behav Brain Res 296:30–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Coric V, Feldman HH, Oren DA, Shekhar A, Pultz J, Dockens RC, Wu X, Gentile KA, Huang S-P, Emison E, Delmonte T, D’Souza BB, Zimbroff DL, Grebb JA, Goddard AW, Stock EG (2010) Multicenter, randomized, double-blind, active comparator and placebo-controlled trial of a corticotropin-releasing factor receptor-1 antagonist in generalized anxiety disorder. Depress Anxiety 27:417–425. [DOI] [PubMed] [Google Scholar]
  19. De Souza EB, Insel TR, Perrin MH, Rivier J, Vale WW, Kuhar MJ (1985) Corticotropin-releasing factor receptors are widely distributed within the rat central nervous system: an autoradiographic study. J Neurosci Off J Soc Neurosci 5:3189–3203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Dixon ML, Fox KCR, Christoff K (2014) Evidence for rostro-caudal functional organization in multiple brain areas related to goal-directed behavior. Brain Res 1572:26–39. [DOI] [PubMed] [Google Scholar]
  21. Franowicz JS, Arnsten AFT (1998) The α-2a noradrenergic agonist, guanfacine, improves delayed response performance in young adult rhesus monkeys. Psychopharmacology (Berl) 136:8–14. [DOI] [PubMed] [Google Scholar]
  22. Fuster J (2015) The Prefrontal Cortex. Academic Press. [Google Scholar]
  23. Gabbott PLA, Warner TA, Jays PRL, Salway P, Busby SJ (2005) Prefrontal cortex in the rat: Projections to subcortical autonomic, motor, and limbic centers. J Comp Neurol 492:145–177. [DOI] [PubMed] [Google Scholar]
  24. Gamo NJ, Wang M, Arnsten AFT (2010) Methylphenidate and Atomoxetine Enhance Prefrontal Function Through α2-Adrenergic and Dopamine D1 Receptors. J Am Acad Child Adolesc Psychiatry 49:1011–1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gescheider GA (1985) Psychophysics: method, theory, and application. L. Erlbaum Associates. [Google Scholar]
  26. Grissom NM, Reyes TM (2019) Let’s call the whole thing off: evaluating gender and sex differences in executive function. Neuropsychopharmacology 44:86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Gritton HJ, Howe WM, Mallory CS, Hetrick VL, Berke JD, Sarter M (2016) Cortical cholinergic signaling controls the detection of cues. Proc Natl Acad Sci 113:E1089–E1097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Haber SN (2003) The primate basal ganglia: parallel and integrative networks. J Chem Neuroanat 26:317–330. [DOI] [PubMed] [Google Scholar]
  29. Hoover WB, Vertes RP (2007) Anatomical analysis of afferent projections to the medial prefrontal cortex in the rat. Brain Struct Funct 212:149–179. [DOI] [PubMed] [Google Scholar]
  30. Howerton AR, Roland AV, Fluharty JM, Marshall A, Chen A, Daniels D, Beck SG, Bale TL (2014) Sex Differences in Corticotropin-Releasing Factor Receptor-1 Action Within the Dorsal Raphe Nucleus in Stress Responsivity. Biol Psychiatry 75:873–883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hupalo S, Berridge CW (2016) Working Memory Impairing Actions of Corticotropin-Releasing Factor (CRF) Neurotransmission in the Prefrontal Cortex. Neuropsychopharmacology 41:2733–2740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hupalo S, Bryce CA, Bangasser DA, Berridge CW, Valentino RJ, Floresco SB (2019a) Corticotropin-Releasing Factor (CRF) circuit modulation of cognition and motivation. Neurosci Biobehav Rev 103:50–59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Hupalo S, Martin AJ, Green RK, Devilbiss DM, Berridge CW (2019b) Prefrontal Corticotropin-Releasing Factor (CRF) Neurons Act Locally to Modulate Frontostriatal Cognition and Circuit Function. J Neurosci 39:2080–2090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Itoga CA, Chen Y, Fateri C, Echeverry PA, Lai JM, Delgado J, Badhon S, Short A, Baram TZ, Xu X (2019) New viral-genetic mapping uncovers an enrichment of corticotropin-releasing hormone-expressing neuronal inputs to the nucleus accumbens from stress-related brain regions. J Comp Neurol. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kehne JH, Cain CK (2010) Therapeutic utility of non-peptidic CRF1 receptor antagonists in anxiety, depression, and stress-related disorders: evidence from animal models. Pharmacol Ther 128:460–487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Koob GF, Zorrilla EP (2012) Update on Corticotropin-Releasing Factor Pharmacotherapy for Psychiatric Disorders: A Revisionist View. Neuropsychopharmacology 37:308–309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kubota Y (2014) Untangling GABAergic wiring in the cortical microcircuit. Curr Opin Neurobiol 26:7–14. [DOI] [PubMed] [Google Scholar]
  38. Kwako LE, Spagnolo PA, Schwandt ML, Thorsell A, George DT, Momenan R, Rio DE, Huestis M, Anizan S, Concheiro M, Sinha R, Heilig M (2015) The Corticotropin Releasing Hormone-1 (CRH1) Receptor Antagonist Pexacerfont in Alcohol Dependence: A Randomized Controlled Experimental Medicine Study. Neuropsychopharmacology 40:1053–1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lapiz MDS, Morilak DA (2006) Noradrenergic modulation of cognitive function in rat medial prefrontal cortex as measured by attentional set shifting capability. Neuroscience 137:1039–1049. [DOI] [PubMed] [Google Scholar]
  40. Mailly P, Aliane V, Groenewegen HJ, Haber SN, Deniau J-M (2013) The Rat Prefrontostriatal System Analyzed in 3D: Evidence for Multiple Interacting Functional Units. J Neurosci 33:5718–5727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. McGaughy J, Kaiser T, Sarter M (1996) Behavioral vigilance following infusions of 192 IgG-saporin into the basal forebrain: selectivity of the behavioral impairment and relation to cortical AChE-positive fiber density. Behav Neurosci 110:247–265. [DOI] [PubMed] [Google Scholar]
  42. McGaughy J, Sarter M (1995) Behavioral vigilance in rats: task validation and effects of age, amphetamine, and benzodiazepine receptor ligands. Psychopharmacology (Berl) 117:340–357. [DOI] [PubMed] [Google Scholar]
  43. McGaughy J, Sarter M (1999) Effects of ovariectomy, 192 IgG-saporin-induced cortical cholinergic deafferentation, and administration of estradiol on sustained attention performance in rats. Behav Neurosci 113:1216–1232. [DOI] [PubMed] [Google Scholar]
  44. Meng Q-Y, Chen X-N, Tong D-L, Zhou J-N (2011) Stress and glucocorticoids regulated corticotropin releasing factor in rat prefrontal cortex. Mol Cell Endocrinol 342:54–63. [DOI] [PubMed] [Google Scholar]
  45. Merchenthaler I (1984) Corticotropin releasing factor (CRF)-like immunoreactivity in the rat central nervous system. Extrahypothalamic distribution. Peptides 5, Supplement 1:53–69. [DOI] [PubMed] [Google Scholar]
  46. Millan MJ et al. (2012) Cognitive dysfunction in psychiatric disorders: characteristics, causes and the quest for improved therapy. Nat Rev Drug Discov 11:141–168. [DOI] [PubMed] [Google Scholar]
  47. Million M, Grigoriadis DE, Sullivan S, Crowe PD, McRoberts JA, Zhou H, Saunders PR, Maillot C, Mayer EA, Taché Y (2003) A novel water-soluble selective CRF1 receptor antagonist, NBI 35965, blunts stress-induced visceral hyperalgesia and colonic motor function in rats. Brain Res 985:32–42. [DOI] [PubMed] [Google Scholar]
  48. Pezze M, McGarrity S, Mason R, Fone KC, Bast T (2014) Too Little and Too Much: Hypoactivation and Disinhibition of Medial Prefrontal Cortex Cause Attentional Deficits. J Neurosci 34:7931–7946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Robinson SL, Perez-Heydrich CA, Thiele TE (2019) Corticotropin Releasing Factor Type 1 and 2 Receptor Signaling in the Medial Prefrontal Cortex Modulates Binge-Like Ethanol Consumption in C57BL/6J Mice. Brain Sci 9:171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rueckert L, Grafman J (1996) Sustained attention deficits in patients with right frontal lesions. Neuropsychologia 34:953–963. [DOI] [PubMed] [Google Scholar]
  51. Sarter M, Givens B, Bruno JP (2001) The cognitive neuroscience of sustained attention: where top-down meets bottom-up. Brain Res Rev 35:146–160. [DOI] [PubMed] [Google Scholar]
  52. Sarter M, McGaughy J (1998) Assessment of sustained and divided attention in rats: aspects of validity. Psychopharmacology (Berl) 138:260–262. [DOI] [PubMed] [Google Scholar]
  53. Sauvage M, Steckler T (2001) Detection of corticotropin-releasing hormone receptor 1 immunoreactivity in cholinergic, dopaminergic and noradrenergic neurons of the murine basal forebrain and brainstem nuclei – potential implication for arousal and attention. Neuroscience 104:643–652. [DOI] [PubMed] [Google Scholar]
  54. Schmitt LI, Wimmer RD, Nakajima M, Happ M, Mofakham S, Halassa MM (2017) Thalamic amplification of cortical connectivity sustains attentional control. Nature 545:219–223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Schreiber AL, Lu Y-L, Baynes BB, Richardson HN, Gilpin NW (2017) Corticotropin-releasing factor in ventromedial prefrontal cortex mediates avoidance of a traumatic stress-paired context. Neuropharmacology 113:323–330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Schwandt ML, Cortes CR, Kwako LE, George DT, Momenan R, Sinha R, Grigoriadis DE, Pich EM, Leggio L, Heilig M (2016) The CRF1 Antagonist Verucerfont in Anxious Alcohol-Dependent Women: Translation of Neuroendocrine, But not of Anti-Craving Effects. Neuropsychopharmacology 41:2818–2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Senut MC, Menetrey D, Lamour Y (1989) Cholinergic and peptidergic projections from the medial septum and the nucleus of the diagonal band of broca to dorsal hippocampus, cingulate cortex and olfactory bulb: A combined wheatgerm agglutinin-apohorseradish peroxidase-gold immunohistochemical study. Neuroscience 30:385–403. [DOI] [PubMed] [Google Scholar]
  58. Sesack SR, Deutch AY, Roth RH, Bunney BS (1989) Topographical organization of the efferent projections of the medial prefrontal cortex in the rat: An anterograde tract-tracing study with Phaseolus vulgaris leucoagglutinin. J Comp Neurol 290:213–242. [DOI] [PubMed] [Google Scholar]
  59. Spencer RC, Berridge CW (2019) Receptor and circuit mechanisms underlying differential procognitive actions of psychostimulants. Neuropsychopharmacology:1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Spencer RC, Devilbiss DM, Berridge CW (2015) The Cognition-Enhancing Effects of Psychostimulants Involve Direct Action in the Prefrontal Cortex. Biol Psychiatry 77:940–950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Spencer RC, Klein RM, Berridge CW (2012) Psychostimulants Act Within the Prefrontal Cortex to Improve Cognitive Function. Biol Psychiatry 72:221–227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Spierling SR, Zorrilla EP (2017) Don’t stress about CRF: assessing the translational failures of CRF1 antagonists. Psychopharmacology (Berl) 234:1467–1481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Swanson LW, Sawchenko PE, Rivier J, Vale WW (1983) Organization of Ovine Corticotropin-Releasing Factor Immunoreactive Cells and Fibers in the Rat Brain: An Immunohistochemical Study. Neuroendocrinology 36:165–186. [DOI] [PubMed] [Google Scholar]
  64. Tanila H, Rämä P, Carlson S (1996) The effects of prefrontal intracortical microinjections of an alpha-2 agonist, alpha-2 antagonist and lidocaine on the delayed alternation performance of aged rats. Brain Res Bull 40:117–119. [DOI] [PubMed] [Google Scholar]
  65. Thiebaut de Schotten M, Urbanski M, Batrancourt B, Levy R, Dubois B, Cerliani L, Volle E (2017) Rostro-caudal Architecture of the Frontal Lobes in Humans. Cereb Cortex 27:4033–4047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Uribe-Mariño A, Gassen NC, Wiesbeck MF, Balsevich G, Santarelli S, Solfrank B, Dournes C, Fries GR, Masana M, Labermeier C, Wang X-D, Hafner K, Schmid B, Rein T, Chen A, Deussing JM, Schmidt MV (2016) Prefrontal Cortex Corticotropin-Releasing Factor Receptor 1 Conveys Acute Stress-Induced Executive Dysfunction. Biol Psychiatry 80:743–753. [DOI] [PubMed] [Google Scholar]
  67. Valentino RJ, Reyes B, Van Bockstaele E, Bangasser D (2012) Molecular and cellular sex differences at the intersection of stress and arousal. Neuropharmacology 62:13–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Valera EM, Brown A, Biederman J, Faraone SV, Makris N, Monuteaux MC, Whitfield-Gabrieli S, Vitulano M, Schiller M, Seidman LJ (2010) Sex Differences in the Functional Neuroanatomy of Working Memory in Adults With ADHD. Am J Psychiatry 167:86–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Van Pett K, Viau V, Bittencourt JC, Chan RKW, Li H-Y, Arias C, Prins GS, Perrin M, Vale W, Sawchenko PE (2000) Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse. J Comp Neurol 428:191–212. [DOI] [PubMed] [Google Scholar]
  70. Vogelsang DA, D’Esposito M (2018) Is There Evidence for a Rostral-Caudal Gradient in Fronto-Striatal Loops and What Role Does Dopamine Play? Front Neurosci 12 Available at: https://www.frontiersin.org/articles/10.3389/fnins.2018.00242/full [Accessed December 14, 2020]. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Voorn P, Vanderschuren LJMJ, Groenewegen HJ, Robbins TW, Pennartz CMA (2004) Putting a spin on the dorsal–ventral divide of the striatum. Trends Neurosci 27:468–474. [DOI] [PubMed] [Google Scholar]
  72. Yarur HE, Vega-Quiroga I, González MP, Noches V, Thomases DR, Andrés ME, Ciruela F, Tseng KY, Gysling K (2020) Inhibitory Control of Basolateral Amygdalar Transmission to the Prefrontal Cortex by Local Corticotrophin Type 2 Receptor. Int J Neuropsychopharmacol 23:108–116. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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