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. Author manuscript; available in PMC: 2025 Apr 23.
Published in final edited form as: Eur J Neurosci. 2019 Feb 15;50(1):1712–1726. doi: 10.1111/ejn.14355

Selective vulnerability of dorsal raphe-medial prefrontal cortex projection neurons to corticosterone-induced hypofunction

Eric W Prouty 1, Daniel J Chandler 2, Wen-Jun Gao 1, Barry D Waterhouse 2
PMCID: PMC12017375  NIHMSID: NIHMS2075233  PMID: 30687960

Abstract

Glucocorticoid hormones and serotonin (5-HT) are strongly associated with the development and treatment of depression, respectively. Glucocorticoids regulate the function of serotonergic neurons in the dorsal raphe nucleus (DR), which are the major source of 5-HT to the forebrain. DR 5-HT neurons are electrophysiologically heterogeneous, though whether this phenotypic variation aligns with specific brain functions or neuropsychiatric disease states is largely unknown. The goal of this work was to determine if chronic exogenous glucocorticoid administration differentially affects the electrophysiological profile of DR neurons implicated in the regulation of emotion versus visual sensation by comparing properties of cells projecting to medial prefrontal cortex (mPFC) versus lateral geniculate nucleus (LGN). Following retrograde tracer injection into mPFC or LGN, male Sprague-Dawley rats received daily injections of corticosterone (CORT) for 21 days, after which whole-cell patch clamp recordings were made from retrogradely labeled DR neurons. CORT-treatment significantly increased the action potential half-width of LGN-projecting DR neurons, but did not significantly affect the firing frequency or excitatory postsynaptic currents of these cells. CORT-treatment significantly reduced the input resistance, evoked firing frequency, and spontaneous excitatory postsynaptic current frequency of mPFC-projecting DR neurons, indicating a concurrent reduction of both intrinsic excitability and excitatory drive. Our results suggest that the serotonergic regulation of cognitive and emotional networks in the mPFC may be more sensitive to the effects of glucocorticoid excess than visual sensory circuits in the LGN and that reduced 5-HT transmission in the mPFC may underlie the association between glucocorticoid excess and depression.

Keywords: lateral geniculate nucleus, retrograde tracing, Sprague-Dawley rat, whole-cell patch clamp electrophysiology

1 |. INTRODUCTION

Glucocorticoids are steroid hormones which facilitate and synchronize the physiological and behavioral responses to a stressor (Chrousos, 2016). The primary glucocorticoids in humans and rodents are cortisol and corticosterone (CORT), respectively (Oster et al., 2017). While glucocorticoids are vital for maintaining homeostasis acutely, prolonged elevations of these hormones are associated with neuropsychiatric dysfunction, particularly depression (Gold, 2015; McEwen et al., 2015; Vreeburg et al., 2009). The prevalence of major depressive disorder (MDD) in patients with Cushing syndrome, a condition resulting from glucocorticoid excess, is 50%–80% (Pivonello et al., 2015; Starkman, 2013), compared to 4.4% in the general population (Ferrari et al., 2013). Notably, the degree of cortisol normalization in patients with Cushing syndrome after treatment is correlated with the degree of improvement in depressive symptoms (Pivonello et al., 2015; Starkman, 2013). Excess cortisol and disruption of its normal circadian rhythm are some of the most commonly observed clinical features in depressed patients (Juruena, Bocharova, Agustini, & Young, 2018; Pariante & Lightman, 2008). The enormous public health burden of MDD (Ferrari et al., 2013) and the widespread use of high-dose glucocorticoid therapies in the US (Gray, Kogan, Marrocco, & McEwen, 2017) indicate that the identification and characterization of specific neural pathways underlying the associations between glucocorticoid abnormalities and depressive symptoms may have significant clinical impact.

The interaction of glucocorticoids and the monoamine neurotransmitter serotonin (5-HT) is of particular interest because nearly all FDA-approved antidepressant medications target 5-HT transmission (Artigas, 2013). Mounting evidence describing developmental, molecular, and electrophysiological heterogeneity of brainstem 5-HT neurons has led to the idea that serotonergic regulation of specific brain functions results from subsets of 5-HT neurons which can differ with respect to their afferent input, intrinsic properties, and specific efferent targets (Commons, 2016; Fernandez et al., 2016; Gaspar & Lillesaar, 2012; Okaty et al., 2015). Serotonergic neurons of the dorsal raphe nucleus (DR) are the major source of 5-HT to the forebrain and project to many limbic and cortical structures implicated in neuropsychiatric disorders (Andrade & Haj-Dahmane, 2013; Calizo et al., 2011; Hale & Lowry, 2011; Muzerelle, Scotto-Lomassese, Bernard, Soiza-Reilly, & Gaspar, 2016; Vertes, 1991). Glucocorticoids regulate the output of DR 5-HT neurons by influencing cellular functions including 5-HT synthesis and electrophysiology (Jochems et al., 2015; Morimoto, Morita, Ozawa, Yokoyama, & Kawata, 1996). CORT inhibits tryptophan hydroxylase and leads to circadian variation of 5-HT synthesis and release in rodents (Fuxe et al., 1987; Malek, Sage, Pevet, & Raison, 2007; Morimoto et al., 1996). In slice physiology experiments, bath application of CORT has been shown to reduce presynaptic glutamate release onto DR 5-HT neurons (Wang, Shen, & Haj-Dahmane, 2012) and DR 5-HT neurons from mice injected with dexamethasone displayed reduced current-evoked firing (Espallergues et al., 2012). Rodent studies have shown that deletion or inhibition of the glucocorticoid receptor (GR) in DR neurons result in anxiolytic (Vincent & Jacobson, 2014) and antidepressant (Jochems et al., 2015) behavioral responses, but whether functionally specific subsets of DR neurons are differentially affected by chronic glucocorticoid excess has not been explored.

In rodents, chronic injections of high-dose CORT result in prolonged exposure to elevated CORT levels, abolishment of the circadian CORT rhythm, and depression-like behaviors (Kott, Mooney-Leber, Shoubah, & Brummelte, 2016; Li et al., 2015; Marks, Fournier, & Kalynchuk, 2009). We examined the effects of chronic CORT injections on the electrophysiological properties of DR neurons projecting to the medial prefrontal cortex (mPFC) or the lateral geniculate nucleus (LGN) of the thalamus. mPFC functions include working memory, decision making, and top-down regulation of emotion (Dalley, Cardinal, & Robbins, 2004; Quirk & Beer, 2006), while LGN functions include the relay of visual sensory information to the primary visual cortex, as well as to oculomotor, circadian, and vestibular centers (Monavarfeshani, Sabbagh, & Fox, 2017; Sherman & Koch, 1986).

mPFC cytoarchitecture, connectivity, and 5-HT receptor expression suggest that reductions in prefrontal 5-HT could result in symptoms characteristic of MDD (Anderson et al., 2016; Duman, Aghajanian, Sanacora, & Krystal, 2016; Heller, 2016; Mengod, Palacios, & Cortes, 2015; Pivonello et al., 2015). In human imaging studies, depressed subjects often show anatomical or metabolic signs of mPFC dysfunction (Drevets, Price, & Furey, 2008; Fales et al., 2008; Siegle, Thompson, Carter, Steinhauer, & Thase, 2007). mPFC pyramidal neurons expressing the 5-HT2A receptor are implicated in the regulation of negative emotional processing in the amygdala and decreased serotonergic excitation of this descending pathway may contribute to amygdala hyperactivity and feelings of hopelessness in depressed and suicidal patients (Aznar & Klein, 2013; Fisher et al., 2009; van Heeringen et al., 2003; Lemogne, Delaveau, Freton, Guionnet, & Fossati, 2012; Phillips et al., 2015). We hypothesized that mPFC-projecting DR neurons from CORT-treated rats would display an electrophysiological profile indicative of hypofunction relative to those from vehicle-treated rats. While glucocorticoid abnormalities have been associated with deficits in visual memory tasks among depressed patients, these are believed to result from cortical dysfunction and decrements in attention and working memory, not visual sensory pathways (Pivonello et al., 2015; Zaninotto et al., 2016). Therefore, we expected that the effects of CORT-treatment on LGN-projecting neurons would be indicative of less functional change than those observed in mPFC-projecting cells.

2 |. MATERIALS AND METHODS

2.1 |. Subjects

The Drexel University College of Medicine Institutional Animal Care and Use Committee approved all animal procedures and protocols. Male Sprague-Dawley rats were acquired from Taconic Biosciences (Hudson, NY). Rats were group housed (2–4 per cage) in a reverse light cycle (lights off at 7:00 a.m., lights on at 7:00 p.m.) with ad libidum access to food and water. A total of 75 rats were used in these experiments (sucrose preference testing, n = 16; electrophysiology, n = 59).

2.2 |. Stereotaxic surgery

As described in (Prouty, Chandler, & Waterhouse, 2017). Young adult (P56, ~250 g) rats received stereotaxic injections of Fluoro-Gold (FG, Fluorochrome Inc., Denver, CO) into the mPFC or LGN. Anesthesia was induced and maintained via isoflurane inhalation at concentrations of 4% and 2.5% isoflurane (v/v), respectively, in 95% oxygen and 5% carbon dioxide. Throughout each surgical procedure, body temperature was monitored and maintained at 37°C using a water-circulating heating pad. Stereotaxic coordinates were based on (Paxinos & Watson, 1997). The following coordinates for each target region are listed relative to bregma in the anteroposterior (AP), mediolateral (ML), and dorsoventral (DV) planes. mPFC coordinates: AP +3.2 mm, ML ±1.3 mm, DV −4.5 mm. LGN coordinates: AP −4.8 mm, ML ±3.9 mm, DV −4.6 mm. DV measurements were made from the skull surface, and when targeting the mPFC, at an angle of 10° from perpendicular to the skull. All injections were made into the left hemisphere. 0.2 μl pressure injections of a 3% FG solution (FG dissolved in distilled water) were made using a Hamilton Neuros syringe (Hamilton Co., Reno, NV) with a 33-gauge stainless steel, non-beveled needle. The syringe was manually dispensed using a Kopf Model 5000 microinjection unit to achieve an injection rate of 0.036 μl/min. The syringe was left in place for 10 min after completion of each injection to allow for tracer diffusion. Rodents received a subcutaneous injection of buprenorphine sustained-release (0.5 mg/kg) approximately 1 hr prior to the end of surgery and 5% lidocaine gel was applied to the incision site following skin closure with wound clips.

2.3 |. Corticosterone injections

The administration of CORT or vehicle injections began 7 days after surgery (animals used for electrophysiology) or 14 days after arrival in the animal facility (animals used for sucrose preference testing). Rats received once-daily subcutaneous injections of either corticosterone or vehicle for 21 days. Injections were timed to coincide with the approximate circadian nadir of the rodents’ circulating corticosterone levels (2–3 hr before lights on), which occurs near the end of their active (dark) phase. Corticosterone (C2505, Sigma-Aldrich, St. Louis, MO) was dissolved in a mixture of 10% ethanol in sesame oil (S3547, Sigma-Aldrich) and injected at a dose of 40 mg/kg and volume of 1 ml/kg. This dosing regimen has been repeatedly shown to increase serum CORT levels and produce metabolic and behavioral effects in rodents (Kott et al., 2016; Sterner & Kalynchuk, 2010). Rat body weight was measured daily. Vehicle injections consisted of 10% ethanol in sesame oil and were also administered at a volume of 1 ml/kg.

In order to examine the impact of chronically elevated CORT levels and disrupted CORT rhythm on sucrose preference and electrophysiology, experiments were conducted 24 hr after the final CORT injection to avoid a direct acute pharmacological effect of CORT. The 24-hr time point was chosen based on studies describing the pharmacokinetics of CORT in rats (Fediuc, Campbell, & Riddell, 2006; Sapolsky, Krey, & McEwen, 1985). As weight loss is a robust indicator of chronically elevated CORT levels in rats (Johnson, Fournier, & Kalynchuk, 2006; Lee et al., 2013; Workman et al., 2016), plasma CORT levels were not obtained at the time of sucrose preference testing or electrophysiology.

2.4 |. Sucrose preference test

Eighteen hours after receiving the final injection of CORT (n = 8) or vehicle (n = 8), rats were individually housed for 48 hr and given equal access to two drinking bottles, one containing 1% sucrose dissolved in tap water and the other containing tap water. The weight of the filled bottles was recorded prior to testing. After 24 hr, the bottle positions were reversed, and after another 24 hr the test was concluded and the bottles were weighed. The difference between pre-test and post-test bottle weight was used to determine the amount of liquid consumed by each animal. To calculate sucrose preference, the amount of sucrose-sweetened water consumed over the 48-hr testing period was divided by the total amount of liquid consumed during that time.

2.5 |. Tissue processing for electrophysiology

Eighteen hours after the last injection of CORT or vehicle, rats were deeply anesthetized with an intraperitoneal injection of Euthasol (100 mg/kg, Virbac, Ft. Worth, TX) and transcardially perfused with 60 ml of ice-cold, carbogen-saturated (95% O2, 5% CO2), protective-slicing artificial cerebrospinal fluid (aCSF) of the following composition, in mM: Choline-Cl 92, KCl 2.5, NaH2PO4 1.2, NaHCO3 30, HEPES 20, CaCl2 0.5, MgSO4 10, d-glucose 25, Na-ascorbate 5, Na-pyruvate 3, thiourea 2 (pH 7.4, 300 mOsm). Coronal sections of brainstem tissue containing dorsal raphe were made at a thickness of 300 μm using a ceramic blade (EF-INZ10, Cadence Inc., Staunton, VA) and Leica VT1200S vibratome, at a speed of 0.06 mm/s and amplitude of 1.00 mm. The tissue sections were placed in a holding chamber filled with aCSF of the following composition, in mM: NaCl 124, KCl 2.5, NaH2PO4 1.25, NaHCO3 24, CaCl2 2.5, MgSO4 2, d-glucose 10 (pH 7.4, 300 mOsm) which was continuously bubbled with carbogen. After 1 hr at 37°C, the holding chamber was kept at room temperature. The protective aCSF and ceramic blade were used to improve the viability of adult tissue sections, as described by (Ting, Daigle, Chen, & Feng, 2014).

2.6 |. Whole-cell patch clamp electrophysiology

Tissue sections were individually transferred to a recording chamber and immersed in a continuous flow of carbogen-saturated aCSF (2 ml/min) maintained at 34°C. Using an Olympus BX51WI microscope and Samsung SCB-2001 camera, individual DR neurons were identified with infrared differential interference contrast and examined for the presence of FG by briefly switching to UV fluorescence. Recordings from FG-positive neurons were made using glass patch pipettes (resistance 5–8 MΩ) filled with an intracellular recording solution composed of the following, in mM: K-gluconate 120, KCl 20, MgCl2 2, HEPES 10, EGTA 0.2, Na2-ATP 2 (pH 7.4, 290 mOSm). After gigaohm seal formation and rupture of the neuronal membrane, whole-cell recordings were obtained using a MultiClamp 700A amplifier, DigiData 1322A digitizer and ClampEx 9.2 software (Molecular Devices, San Jose, CA). If resting membrane potential and series resistance were stable over 2 min, current steps (−100 to 450 pA in 50 pA steps) were applied to the cell to evaluate membrane properties and evoked firing frequency. The recording mode was then switched to voltage clamp and neurons were held at −70 mV to record spontaneous excitatory postsynaptic currents (sEPSCs) for 4 min. At this holding potential the driving force for chloride ions is much smaller than the forces driving cation conductance; therefore, while unlikely, the possibility of a minor chloride-mediated contribution to the recorded inward currents cannot be excluded. Most of the recorded neurons were located in the midline subregions of the DR between 7 and 8 mm caudal to bregma, though 2–3 LGN-projecting cells in each treatment group were located in the lateral wings of the nucleus.

2.7 |. Analysis of electrophysiological data

Recorded data were analyzed using ClampFit 10.2 (Molecular Devices, San Jose, CA). The following properties were assessed in current clamp traces: input resistance (IR), membrane time constant (τ), resting membrane potential (RMP), AP threshold, AP amplitude, AP half-width (AP duration at half-peak voltage), afterhyperpolarization (AHP) amplitude, and AHP half-recovery time (AHP t1/2, time needed to repolarize from AHP peak to half-peak voltage). All properties except AHP amplitude and half-recovery time were measured using the rheobase spike. AHP parameters were measured using the first spike at the current step immediately following the rheobase step (rheobase current +50 pA) so that each trace had more than one spike. Firing frequency at rheobase +50 pA did not significantly differ across groups (F3,39 = 1.994, p = 0.131). Evoked firing frequency was determined by counting the number of APs fired in response to each 1-s current step. In voltage clamp traces, sEPSCs were detected using an automated template-matching protocol. Mean sEPSC amplitude and frequency were calculated across the full duration of the recording for each cell.

2.8 |. Statistical analysis

Statistical analyses were performed with IBM Statistics SPSS version 24 and GraphPad Prism 7. All data sets were tested for normality using the D’Agostino-Pearson test. In order to meet assumptions required for parametric testing, AP half-width values were square root transformed for normality and sucrose preference percentage values were logit transformed to stabilize variance. Independent samples t tests were used to determine the effect of CORT on percent change in bodyweight and sucrose preference. Two-way ANOVAs were performed to determine the effects of projection target (mPFC vs. LGN), treatment (vehicle vs. CORT), and interaction (projection target × treatment) on all parameters except evoked firing frequency. We identified two a priori comparisons that reflected our goal of determining the effect of CORT on target-specified populations of DR projection neurons: (a) mPFC-projecting neurons from animals treated with vehicle versus CORT and (b) LGN-projecting neurons from animals treated with vehicle versus CORT. These comparisons were conducted if a significant interaction was identified using two-way ANOVA. A mixed-model ANOVA was used to determine the effect of treatment (vehicle vs. CORT), injected current (within-subjects factor with nine levels: 50–450 pA), and interaction (treatment × current) on AP firing frequency. The Greenhouse-Geisser correction was used to account for violations of sphericity. Significant interactions were followed by post-hoc t tests to determine the effect of treatment at each level of current. The Bonferroni method was applied to planned and post-hoc comparisons to control for familywise error rate. GraphPad Prism 7 was used to generate all graphs. Data in all figures are presented as mean ± SEM unless otherwise indicated.

3 |. RESULTS

3.1 |. Bodyweight and sucrose preference

The experimental timeline of animals used for sucrose preference testing is shown in Figure 1a. Weight loss is a robust metabolic consequence of the high dose CORT administration paradigm (Sterner & Kalynchuk, 2010). Rodent bodyweight was measured to confirm that the CORT injections were having a physiological effect. Change in bodyweight resulting from CORT or vehicle injections was consistent across animals receiving the same type of injection. Figure 1b shows the average percent change in bodyweight pooled across all vehicle-treated animals (n = 28) and CORT-treated animals (n = 31). Treatment had a significant effect on bodyweight (t57 = 36.1, p < 0.0001). To confirm that the CORT injections induced a depressive phenotype in rats, we examined the effect of treatment on sucrose preference (Figure 1c). When compared with vehicle-treated animals, CORT-treated animals displayed a significantly reduced preference for sucrose-sweetened drinking water (t14 = 3.86, p = 0.0018).

FIGURE 1.

FIGURE 1

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Chronic injections of corticosterone (CORT) reduce body weight and sucrose preference. (a) Timeline of behavioral experiments. Acclimation = rats were gently handled for 15 min per day in their home cage to desensitize them to the experimenter. SP = sucrose preference test. (b) Treatment (vehicle vs. CORT) had a significant effect on rodent body weight (t57 = 36.1, p < 0.0001). Animals receiving vehicle injections showed a 28% increase in body weight over the 21-day administration period, while animals receiving CORT injections showed a 13% decrease. (c) CORT injections were associated with a significant reduction in sucrose preference (t14 = 3.86, p = 0.0018). Sucrose-sweetened water accounted for 95% of the total liquid consumed by vehicle-treated animals during the 48-hr testing period, but only 80% of the total liquid consumed by CORT-treated animals. *p < 0.05

3.2 |. Passive and active membrane properties

The timeline for electrophysiological experiments is shown in Figure 2a. The retrograde tracer FG was used to label DR neurons projecting to mPFC or LGN. Representative injections of FG into the mPFC and LGN are shown in Figure 2b,c, respectively. Figure 3a shows a typical action potential waveform recorded from a FG-labeled DR neuron. Representative action potential waveforms from each group are shown in Figure 3b and traces used to calculate input resistance are shown in Figure 3c. To determine if chronic CORT injections affected the intrinsic membrane properties of DR projection neurons, the following four groups of cells were compared: mPFC-projecting neurons from vehicle-treated animals (n = 10 cells from six animals), mPFC-projecting neurons from CORT-treated animals (n = 10 cells from seven animals), LGN-projecting neurons from vehicle-treated animals (n = 13 cells from nine animals), and LGN-projecting cells from CORT-treated animals (n = 10 cells from eight animals). The interaction of projection target and treatment had a significant effect on input resistance (F1,39 = 4.459, p = 0.0412) and planned comparisons revealed that CORT-treatment was associated with a significant decrease in the input resistance of mPFC-projecting DR neurons (t18 = 2.511, p = 0.0218), but not in LGN-projecting neurons (t21 = 0.3472, p = 0.7319; Figure 3d). The main effect of treatment on membrane τ was significant (F1,39 = 5.773, p = 0.0211; Figure 3e). The interaction of projection target and treatment had a significant effect on action potential half-width (F1,39 = 11.409, p = 0.002). Planned comparisons revealed CORT-treatment resulted in a significant increase in AP half-width among LGN-projecting neurons (t21 = 2.845, p = 0.010), but not among mPFC-projecting neurons (t18 = 1.992, p = 0.062; Figure 3f). The effects of CORT on passive and active membrane properties are summarized in Table 1, rows 1–3.

FIGURE 2.

FIGURE 2

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Timeline of electrophysiological experiments and verification of tracer injection sites. (a) Experimental timeline. Acclimation = rats were gently handled for 15 min per day in their home cage to desensitize them to the experimenter. Surgery = stereotaxic injection of Fluoro-Gold (FG) into the medial prefrontal cortex (mPFC) or lateral geniculate nucleus (LGN), Ephys = whole-cell patch clamp recordings from FG-labeled neurons in the dorsal raphe nucleus. Photomicrographs of representative FG injections in the mPFC (b) and LGN (c) with corresponding stereotaxic atlas images showing the location of target sites (Paxinos & Watson, 1997). FG fluorescence (yellow) indicates the location of deposited tracer. Scale bar = 1 mm.

FIGURE 3.

FIGURE 3

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The effect of chronic corticosterone (CORT) injections on the intrinsic membrane properties of dorsal raphe (DR) projection neurons. (a) Trace of an individual action potential waveform showing how several of the spike properties were measured. AHP amp = afterhyperpolarization amplitude. (b) Traces of four representative action potentials (APs) grouped by the projection target (mPFC vs. LGN) of the recorded neuron. AP half-widths of representative traces (ms): mPFC-projecting = 2.4 (vehicle), 2.3 (CORT) and LGN-projecting = 2.5 (vehicle), 3.5 (CORT). (c) Representative traces in response to 100 pA hyperpolarizing current which are reflective of input resistance (IR). IR of representative traces (MΩ): mPFC-projecting = 377.8 (vehicle), 233.5 (CORT) and LGN-projecting = 400.9 (vehicle), 402.8 (CORT). (d) The interaction of projection target and treatment (vehicle vs. CORT) had a significant effect on IR (F1,39 = 4.46, p = 0.0412). Within the mPFC-projecting population, neurons from CORT-treated animals had significantly lower IR than neurons from vehicle-treated animals (t18 = 2.511, p = 0.0218). (e) Treatment had a significant effect on membrane time constant (F1,39 = 5.773, p = 0.0211), but the effect of target (F1,39 = 0.3093, p = 0.5813) and the interaction of target and treatment (F1,39 = 2.533, p = 0.1195) were not significant. (f) The interaction of projection target and treatment had a significant effect on AP-half width (F1,39 = 11.41, p = 0.002). Within the LGN-projecting population, neurons from CORT-treated animas had significantly greater AP half-width than neurons from vehicle-treated animals (t21 = 2.845, p = 0.010). No significant differences were observed in: (g) resting membrane potential (target F1,39 = 0.430, p = 0.516; treatment F1,39 = 2.490, p = 0.123; interaction F1,39 = 1.776, p = 0.190). (h) AP threshold (target F1,39 = 0.051, p = 0.822; treatment F1,39 = 0.078, p = 0.781; interaction F1,39 = 1.557, p = 0.220). (i) AP amplitude (target F1,39 = 0.397, p = 0.532; treatment F1,39 = 3.959, p = 0.054; interaction F1,39 = 0.073, p = 0.789). (j) AHP amplitude (target F1,39 = 0.016, p = 0.899; treatment F1,39 = 0.642, p = 0.428; interaction F1,39 = 0.698, p = 0.409). (k) AHP half-recovery time (target F1,39 = 1.541, p = 0.222; treatment F1,39 = 0.026, p = 0.872, interaction F1,39 = 1.257, p = 0.269). Bonferroni-adjusted planned comparisons: *p < 0.025, nsp > 0.025 (non-significant)

TABLE 1.

Summary of electrophysiological parameters showing significant changes in response to chronic corticosterone (CORT) administration

Effect of chronic CORT
DR-mPFC neurons LGN neurons
AP half-width — ↑
Input resistance ↓ —
Membrane time constant ↓ —
Evoked firing frequency ↓ —
sEPSC frequency ↓ —
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3.3 |. Evoked firing frequency

Figure 4a shows representative traces of neuronal discharge in response to a depolarizing current of 300 pA. In mPFC-projecting cells, the interaction of current and treatment had a significant effect on AP frequency (F2.6,46 = 4.247, p = 0.01351). Post-hoc comparisons revealed that mPFC-projecting neurons from CORT-treated animals fired significantly fewer APs in response to depolarizing current than those from vehicle-treated animals (Figure 4b). Among LGN-projecting DR neurons, the amount of injected current had a significant effect on AP frequency (F2.3,48 = 43.633, p < 0.0001; Figure 4c), while CORT-treatment and the interaction of current and treatment did not significantly impact the firing rate of those cells.

FIGURE 4.

FIGURE 4

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Chronic corticosterone (CORT) injections decrease the excitability of dorsal raphe (DR) neurons projecting to the medial prefrontal cortex (mPFC). (a) Representative traces showing neuronal discharge in response to a 1 s stimulation of 300 pA depolarizing current. (b) In mPFC-projecting DR neurons, the interaction of current and treatment had a significant effect on action potential (AP) firing frequency (F2.6,46 = 4.25, p = 0.014). Neurons from CORT-treated animals fired significantly fewer APs than neurons from vehicle-treated animals in response to all levels of depolarizing current (50–450 pA). (c) In LGN-projecting neurons, current had a significant effect on AP firing frequency (F2.3,48 = 43.633, p < 0.0001), but the effect of treatment (F1,21 = 1.752, p = 0.200) and the interaction of current and treatment (F2.3,48 = 1.486, p = 0.235) were not significant. Bonferroni-adjusted post-hoc comparisons: *p < 0.0056

3.4 |. Spontaneous excitatory postsynaptic currents

To determine if chronic CORT injections affected excitatory synaptic transmission in DR projection neurons, sEPSC amplitude and frequency were compared among the following groups of cells: mPFC-projecting neurons from vehicle-treated animals (n = 10 cells from six animals), mPFC-projecting neurons from CORT-treated animals (n = 14 cells from nine animals), LGN-projecting neurons from vehicle-treated animals (n = 12 cells from nine animals), and LGN-projecting cells from CORT-treated animals (n = 11 cells from nine animals). Representative voltage clamp traces are shown in Figure 5a. No significant effects of projection target, CORT-treatment, or injection × treatment were observed on sEPSC amplitude (Figure 5b). In contrast, the interaction of projection target and treatment had a significant effect on sEPSC frequency (F1,43 = 10.327, p = 0.0025). Planned comparisons revealed that CORT treatment resulted in a significant decrease in sEPSC frequency among mPFC-projecting neurons (t22 = 5.198, p = 0.0001), but not LGN-projecting neurons (t21 = 0.294, p = 0.772; Figure 5c).

FIGURE 5.

FIGURE 5

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Chronic injections of corticosterone (CORT) reduce the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in dorsal raphe (DR) neurons projecting to medial prefrontal cortex (mPFC). (a) Representative voltage clamp traces from DR neurons held at −70 mV. sEPSC amplitude of representative traces (pA): mPFC-projecting 25.06 (vehicle), 24.84 (CORT) and LGN-projecting 22.95 (vehicle), 28.32 (CORT). sEPSC frequency of representative traces (Hz): mPFC-projecting 9.2 (vehicle), 3.3 (CORT) and LGN-projecting 9.6 (vehicle), 9.8 (CORT). (b) Projection target (F2.6,46 = 4.25, p = 0.014), treatment (F2.6,46 = 4.25, p = 0.014), and the interaction of target and treatment (F2.6,46 = 4.25, p = 0.014) did not have significant effects on sEPSC amplitude. (c) The interaction of projection target and treatment had a significant effect on sEPSC frequency (F1,43 = 10.33, p = 0.0025). CORT treatment resulted in a significant decrease in sEPSC frequency among mPFC-projecting neurons (t22 = 5.198, p = 0.0001), but not LGN-projecting neurons (t21 = 0.383, p = 0.772). Bonferroni-adjusted planned comparisons: *p < 0.025, nsp > 0.025 (non-significant)

4 |. DISCUSSION

The major finding of this work was that exogenous glucocorticoid administration in rats differentially impacted two populations of DR projection neurons based on their efferent targets. Specifically, chronic injections of CORT decreased the intrinsic excitability and excitatory synaptic drive of DR neurons projecting to the mPFC, while having little effect on DR neurons projecting to the LGN (summarized in Table 1). The effects of CORT on the membrane properties of mPFC-projecting neurons suggest that these cells would have a reduced ability to integrate and respond to excitatory afferent input. The decreased responsiveness of these neurons was directly demonstrated by the significant reductions in evoked firing frequency across a wide range depolarizing currents. CORT-treatment also reduced the frequency of sEPSCs in mPFC-projecting DR neurons. The concurrent reduction of excitatory input and intrinsic excitability would likely result in a profound decrease of synaptically driven neuronal firing in vivo and, subsequently, reduced neurotransmitter release within the mPFC. As CORT injections did not produce electrophysiological changes indicative of reduced output in LGN-projecting neurons, our findings suggest that the serotonergic modulation of cognitive and emotional circuitry within the mPFC may be more sensitive to the effects of glucocorticoid excess than 5-HT regulation of visual sensory processing in LGN.

4.1 |. Depressive phenotype of CORT-treated animals

In confirmation of many previous reports, rodents given chronic injections of CORT displayed decreased bodyweight and reduced preference for sucrose-sweetened water (Kott et al., 2016; Marks et al., 2009; Sterner & Kalynchuk, 2010). Reduced sucrose preference is thought to represent a rodent measure of anhedonia (a reduced ability to experience joy or pleasure), which is a core diagnostic criterion of MDD (Kupfer, Frank, & Phillips, 2012; Pizzagalli, 2014). The reductions in bodyweight and sucrose preference resulting from exogenous CORT resemble clinical aspects of MDD—particularly MDD with melancholic features, a subtype of depression characterized by severe anhedonia, weight loss, and a prominent association with excess cortisol (Gold, 2015; Juruena et al., 2018).

4.2 |. Neurochemical identity of recorded neurons

Using the same rat strain, tracer, and stereotaxic procedures, we have previously shown that 90% of 2238 labeled DR-mPFC neurons and 95% of 2117 DR-LGN neurons (total counts from six animals per group) were positive for tryptophan hydroxylase, indicating that both structures receive strong, primarily serotonergic projections from the DR (Prouty et al., 2017). The distribution of labeled cells in the current experiments matched closely with our previous report. DR-mPFC neurons were located within the dorsomedial and ventromedial regions of the DR, predominantly ipsilateral to the injection site. DR-LGN neurons also displayed an ipsilateral predominance with a higher concentration in the lateral wings than in midline regions of the nucleus. The electrophysiological properties of the FG-labeled neurons closely aligned with established properties characteristic of DR serotonergic neurons, including long AP duration and large amplitude AHP with slow recovery time (Calizo et al., 2011; Tuckwell, 2013; Vandermaelen & Aghajanian, 1983). Those signature electrophysiological features in conjunction with our previous neurochemical study showing that the vast majority of DR neurons projecting to mPFC or LGN were tryptophan hydroxylase-positive (Prouty et al., 2017), provide strong support for the putative serotonergic identity of the recorded neurons.

4.3 |. Effects of CORT-treatment on DR neuronal electrophysiology

LGN-projecting cells from CORT-treated animals displayed an increase in AP half-width relative to those from vehicle-treated animals. The increase in AP duration would likely have the most impact at the synaptic terminal, where the duration of depolarization and subsequent calcium influx could affect the timing and amount of neurotransmitter release (McCormick, 2013). As CORT treatment did not have significant effects on the evoked firing frequency or sEPSC frequency of LGN-projecting DR neurons, we suspect that chronic elevations of glucocorticoids would have less impact on DR neuronal output in the LGN than in the mPFC.

In the mPFC-projecting population, neurons from CORT-treated animals had lower input resistance (IR) than neurons from vehicle-treated animals. IR is defined as the change in membrane voltage in response to a given current; the lower IR of CORT-treated, mPFC-projecting DR neurons represents a decrease in neuronal excitability as a greater amount of stimulation would be required to achieve the level of depolarization needed to initiate an AP in those cells (McCormick, 2013). This suggests that in vivo the CORT-treated mPFC-projecting neurons would be less responsive to afferent synaptic input than vehicle-treated neurons. The effect of CORT on the IR of mPFC-projecting neurons may be the result of greater ion channel conductance (McCormick, 2013) and the lack of a concurrent change in RMP suggests that increased chloride conductance may be responsible (Jin et al., 2015; Kirby et al., 2007; Llamosas, Ugedo, & Torrecilla, 2017). GABA-ergic neurons in the periaqueductal gray are important regulators of DR 5-HT activity (Fu et al., 2010; Soiza-Reilly & Commons, 2014). Challis et al. observed decreased excitability in DR 5-HT neurons of mice susceptible to chronic social defeat stress (CSDS); notably, those cells showed greater inhibitory synaptic input from local GABA neurons (Challis et al., 2013). Related studies from that laboratory found that GR signaling within DR 5-HT neurons contributes to the decreased excitability of DR 5-HT cells and social avoidance after CSDS (Espallergues et al., 2012; Jochems et al., 2015). Together those findings suggest that the decreases in input resistance and excitability that we observed in DR-mPFC neurons following chronic CORT exposure could result from increased chloride conductance through GABA receptors and that further exploration of target-specific differences in GABA transmission among DR 5-HT neurons is warranted. We also observed that DR neurons exposed to CORT displayed a smaller membrane time constant (τ) than those exposed to vehicle. This indicates that charge accumulates and dissipates faster in CORT-treated cells, thus shortening the window during which synaptic currents could undergo temporal summation (Byrne, 2013). While the effect of CORT on membrane τ was not target-specific, the in vivo consequences of this effect would likely be more pronounced in mPFC-projecting DR neurons as their reduced ability to integrate synaptic currents would be compounded by decreased membrane responsiveness resulting from lower IR.

In mPFC-projecting DR neurons, CORT-treatment resulted in a profound reduction in evoked firing frequency across a wide range of depolarizing currents (50–450 pA). The decreased responsiveness to current injection is consistent with the CORT-induced changes in passive membrane properties (indicative of reduced excitability) and further supports the idea that DR-mPFC neurons would be less active in vivo. Previous studies have found that DR 5-HT neurons are less responsive to injected current or display reduced firing in vivo after chronic isolation, chronic social defeat, and chronic unpredictable stress (Bambico, Nguyen, & Gobbi, 2009; Espallergues et al., 2012; Sargin, Oliver, & Lambe, 2016). A caveat to the interpretation of our findings is that while exogenous CORT administration models a prominent component of the stress response, it is not equivalent to the experience of stress. The uniform decreases in DR 5-HT neuronal activity described in the above reports may be due to the additive effects of multiple neuroendocrine responses to behavioral stressors (Gray et al., 2017), while the selective decrease in firing frequency that we observed in mPFC-projecting neurons may reflect a more specific role of glucocorticoid signaling pathways.

We also observed a reduction in the frequency of putative AMPA-mediated spontaneous excitatory postsynaptic currents (sEPSCs) in CORT-treated DR neurons projecting to the mPFC, but not in those projecting to the LGN. The amygdala, lateral habenula, lateral hypothalamus, and mPFC are major sources of glutamatergic input to DR (Soiza-Reilly & Commons, 2011; Weissbourd et al., 2014; Zhou et al., 2017). It is possible that the relative contribution of each structure to the excitatory inputs received by DR-mPFC and DR-LGN neurons could differ. Significant differences between the inputs to several projection-specific subpopulations of dopaminergic neurons in the ventral tegmental area (VTA) and substantia nigra have recently been reported (Beier et al., 2015; Lerner et al., 2015; Ogawa & Watabe-Uchida, 2018). Reciprocal connections between mPFC and DR have been implicated in determining responses to stressors and emotional behaviors (Geddes et al., 2016; Volle et al., 2018). Layer 5 pyramidal neurons in PFC are particularly vulnerable to glucocorticoid-induced hypofunction or cell death (Duman et al., 2016; Yuen, Wei, & Yan, 2017); therefore, our sEPSC findings could result from an anatomical bias toward fine reciprocity between DR and mPFC, resulting in greater reductions of glutamatergic input to DR-mPFC neurons than DR-LGN neurons after chronic CORT exposure. While systematic investigations of specific input-output relationships in the serotonergic nuclei have not yet been conducted, advances in viral tracing and genetic markers useable for serotonergic intersectional genetics (Okaty et al., 2015), suggest that such studies will likely be forthcoming (Ogawa & Watabe-Uchida, 2018).

The observed reduction in sEPSC frequency may also result from CORT-induced differences in retrograde signaling by the two groups of projection neurons. Electrophysiological studies have shown DR 5-HT neurons can alter the release probability of glutamate and GABA from presynaptic terminals in the DR using endocannabinoids (eCBs) and nitric oxide (NO) as retrograde messengers (Haj-Dahmane, Beique, & Shen, 2017; Kim et al., 2018; Wang et al., 2012). eCBs have been directly implicated in glucocorticoid-induced reductions of sEPSC frequency in DR 5-HT neurons (Wang et al., 2012) and restraint stress as well as capsaicin injection increase NO production in 5-HT neurons within specific subregions of the nucleus (Nichols et al., 2017; Okere & Waterhouse, 2006a, 2006b). Our previous finding that a significantly greater proportion of DR 5-HT neurons projecting to mPFC versus LGN (60% vs. 22%) have the capacity to synthesize NO also supports the possibility of target-specific differences in retrograde signaling (Prouty et al., 2017). Whether the CORT-induced reduction in sEPSC frequency was the result of retrograde signaling or segregated inputs, the functional outcome would be less excitatory drive to mPFC-projecting DR neurons.

5 |. CONCLUSION

In summary, our findings suggest that mPFC-projecting DR neurons show greater sensitivity to chronic elevations in CORT than LGN-projecting DR neurons, resulting in both intrinsic and synaptic electrophysiological changes indicative of decreased excitability and activity. Therefore, we would expect that 5-HT output in the mPFC of rats receiving chronic injections of CORT would be decreased relative to 5-HT output in the LGN. Within the mPFC, 5-HT signaling impacts the top-down control of subcortical structures such as the VTA and amygdala (Elliott, Tanaka, Schwark, & Andrade, 2018; Mengod et al., 2015; Santana & Artigas, 2017) and dysfunction of those descending projections has been implicated in anhedonia and negative affective bias, respectively (Heller, 2016; Phillips et al., 2015). Deficits in 5-HT transmission within the mPFC have been specifically associated with suicidal intent and negative affective bias in clinical imaging studies (Leyton et al., 2006; Robinson et al., 2013). Notably, increasing 5-HT transmission in mPFC is associated with antidepressant efficacy of selective serotonin reuptake inhibitors (Artigas, Bortolozzi, & Celada, 2018), ketamine (Pham et al., 2017), and deep brain stimulation (Veerakumar et al., 2014) in rodent models.

Our findings suggest that chronic CORT exposure would result in the hypofunction of DR-mPFC neurons, but not DR-LGN neurons; therefore, an important future goal is to determine the impact of chronic CORT on serotonergic modulation of those target circuits, including measurements of extracellular 5-HT and patch-clamp recordings of principal neurons within the mPFC and LGN. Additional future studies could include pharmacological investigation of potential receptors (GR, glutamate, GABA) responsible for the effect of chronic CORT on intrinsic excitability and also how the DR neurons respond to bath application of 5-HT or a 5-HT1A agonist following chronic CORT administration. The latter experiment would provide insight into the effect of glucocorticoid excess on the autoregulation of these target-specific populations and represents an area of particular interest because most antidepressant medications are thought to achieve therapeutic efficacy through accumulation of 5-HT within the DR and desensitization of soma-todendritic 5-HT1A inhibitory autoreceptors on serotonergic projection neurons (Artigas, 2013; Courtney & Ford, 2016).

By linking the differential effects of chronic CORT injections to DR projection neurons with specific anatomical targets, this study serves as an important foundation for understanding how aberrant glucocorticoid signaling may affect 5-HT transmission in specific brain regions. Furthermore, the changes we observed in mPFC-projecting DR neurons after chronic elevations in CORT suggest that reduced 5-HT modulation of mPFC circuits may underlie the prominent association between glucocorticoid excess and depression and that enhanced activity of this pathway may correlate with positive therapeutic responses to serotonergic antidepressants. Further characterization of DR-mPFC neurons, including their specific inputs and molecular profile, may reveal potential targets for selective manipulation of this population to directly examine the role of these neurons in emotional behaviors.

ACKNOWLEDGEMENTS

The authors would like to thank Doug Fox for the histological processing of tissue used to verify tracer injection sites. This research was supported by the National Institute of Mental Health of the National Institutes of Health under award number R01MH101178 to BDW and seed funds from Rowan University School of Osteophathic Medicine to BDW.

Funding information

National Institute of Mental Health of the National Institutes of Health, Grant/Award Number: R01MH101178; Rowan University School of Osteophathic Medicine

Abbreviations:

5-HT

5-hydroxytryptamine

aCSF

artificial cerebrospinal fluid

AHP

afterhyperpolarization

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

CORT

corticosterone

CSDS

chronic social defeat stress

DR

dorsal raphe nucleus

eCB

endocannabinoid

FG

Fluoro-Gold

GABA

γ-aminobutyric acid

GR

glucocorticoid receptor

IR

input resistance

LGN

lateral geniculate nucleus

MDD

major depressive disorder

mPFC

medial prefrontal cortex

NO

nitric oxide

RMP

resting membrane potential

sEPSC

spontaneous excitatory postsynaptic current

VTA

ventral tegmental area

τ

membrane time constant

Footnotes

CONFLICT OF INTEREST

The authors declare no biomedical financial interests or potential conflicts of interest.

DATA ACCESSIBILITY

Requests for supporting data and materials can be sent to the corresponding author.

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