The estrous cycle modulates rat caudate-putamen medium spiny neuron physiology

Abstract

The neuroendocrine environment in which the brain operates is both dynamic and differs by sex. How differences in neuroendocrine state affect neuron properties has been significantly neglected in neuroscience research. Behavioral data across humans and rodents indicate that natural cyclical changes in steroid sex hormone production affect sensorimotor and cognitive behaviors in both normal and pathological contexts. These behaviors are critically mediated by the caudate-putamen. In the caudate-putamen, medium spiny neurons (MSNs) are the predominant and primary output neurons. MSNs express membrane-associated estrogen receptors and demonstrate estrogen sensitivity. However, how the cyclical hormone changes across the estrous cycle may modulate caudate-putamen MSN electrophysiological properties remains unknown. Here, we performed whole-cell patch clamp recordings on male, diestrus female, proestrus female, and estrus female caudate-putamen MSNs. Action potential, passive membrane, and miniature excitatory postsynaptic current properties were assessed. Numerous MSN electrical properties robustly differed by cycle state, including resting membrane potential, rheobase, action potential threshold, maximum evoked action potential firing rate, and inward rectification. Strikingly, when considered independent of estrous cycle phase, all but one of these properties do not significantly differ from male MSNs. These data indicate that female caudate-putamen MSNs are sensitive to the estrous cycle, and more broadly, the importance of considering neuroendocrine state in studies of neuron physiology.

Graphical Abstract

This study demonstrates that caudate-putamen medium spiny neuron excitability differs across the estrous cycle in female rats. Using whole-cell patch clamp electrophysiological assessment, we detected robust changes in numerous electrical properties across the estrous cycle, including resting membrane potential, rheobase, action potential threshold, maximum evoked action potential firing rate, and inward rectification.

Introduction

Patterns of sex steroid hormone production differ markedly between females and males over the lifespan (Gillies & McArthur, 2010). Importantly, adult female humans and rodents, including rats, exhibit cyclical fluctuations in sex steroid hormones including 17β-estradiol (estradiol) and progesterone. In humans, these variations in hormone production occur over the ~28 day menstrual cycle. The human menstrual cycle features the follicular and luteal phases. In rodents, the timing is more rapid, occurring over the ~4–5 day estrous cycle (Becker et al., 2005). The rat estrous cycle phases include the diestrus, proestrus, and estrus phases. Despite the differences in length, there are similarities in the pattern of hormone fluctuation and the associated behavioral changes between humans and rodents. Most research on the adult female hormone cycle has focused on the ovary and associated reproductive functions. However, sex steroid hormones cross the blood brain barrier to act in the central nervous system. Thus, these cyclical neuroendocrine dynamics can potentially modulate neural substrate directly to yield numerous behavioral and neural consequences. Indeed, estrous cycle effects on neuron physiology have been reported in several brain regions (Woolley & McEwen, 1993; Woolley, 1998; Calizo & Flanagan-Cato, 2000; Okamoto et al., 2003; Cooke & Woolley, 2005; Hao et al., 2006; Blume et al., 2017).

Nevertheless, most research has investigated the action of these hormone changes in the context of classical sex-specific reproductive behaviors such as lordosis (Micevych & Meisel, 2017). Numerous other behaviors that may relate to overall reproductive success are also influenced by cycle phase. For example, in both humans and rats, the mid-cycle surge in estradiol is associated with increased locomotor activity (Beatty, 1979; Becker et al., 1987; Smith, 1994; Becker, 2002), improved limb coordination (Becker et al., 1987; Hampson & Kimura, 1988; Hampson, 1990; Jennings et al., 1998; Simic et al., 2010; Zoghi et al., 2015), and in a pathological context, decreased severity of parkinsonism (Quinn & Marsden, 1986; Castrioto et al., 2010). All of these behaviors are mediated by the caudate-putamen, a well-conserved constituent of the basal ganglia present in both rodents and humans that is instrumental for various forms of learning, and sensorimotor performance, among other functions. Acting as a gateway region, the caudate-putamen receives numerous excitatory glutamatergic afferents, including from the cortex and the thalamus, as well as dopaminergic afferents from the substantia nigra (Kreitzer & Malenka, 2008; Palmiter, 2008). These afferents, along with internal circuitry and other inputs, ultimately converge onto the output neuron of the caudate-putamen, the GABAergic medium spiny neuron (MSN).

MSNs comprise ≥95% of the caudate-putamen neuron population. Changes in MSN electrical activity are directly related to changes in behavior (Grillner et al., 2005; Ferguson et al., 2011; Tan et al., 2013). Adult female rat caudate-putamen MSNs express membrane-associated estrogen receptors α, β, and G-protein-coupled receptor 1 (GPER1) (Mermelstein et al., 1996; Schultz et al., 2009; Grove-Strawser et al., 2010; Almey et al., 2012; Almey et al., 2016). MSNs also famously express dopamine receptors, and there are notable differences in both the expression of these receptors and dopamine levels in the caudate-putamen over the estrous cycle in female rats and in response to exogenous estradiol in ovariectomized rats (Di Paolo, 1994; Yoest et al., 2018; Song et al., 2019). Unlike select neuron types in other brain regions, caudate-putamen MSN soma size, density, and overall caudate-putamen volume do not seem to differ by sex (Meitzen et al., 2011; Wong et al., 2016). Thus, research has largely focused on hormone-induced changes in electrophysiological properties (Meitzen et al., 2018). Before puberty, female rat caudate-putamen MSNs exhibit hyperpolarized action potential threshold, decreased afterhyperpolarization magnitude, and increased slope of the evoked action potential to injected positive current curve (Dorris et al., 2015). However, puberty can potentially reorganize neural substrate, including the striatal regions (Juraska et al., 2013; Staffend et al., 2014; Kopec et al., 2018), and introduces new sexually dimorphic hormone dynamics which can alter striatal neuron properties. Likewise, estradiol has also been shown to rapidly modulate L-type calcium channel currents and associated CREB phosphorylation in both prepubertal striatum cell culture and in adult striatum (Mermelstein et al., 1996; Grove-Strawser et al., 2010). In adult rats, in the early 1980s in vivo recordings of spontaneously firing unidentified neurons suggested that caudate-putamen excitability varies across the estrous cycle and in response to estradiol (Arnauld et al., 1981; Tansey et al., 1983). However, for decades it has been unknown whether any cellular electrophysiological property differs by sex or across the adult female hormone cycle in an identified caudate-putamen neuron type. Based upon this literature, we suspected that MSN physiology would be sensitive to the estrous cycle. To interrogate the contributions of estrous cycle and more broadly biological sex, we thus performed whole-cell patch clamp recordings on male, diestrus female, proestrus female, and estrus female rat caudate-putamen MSNs.

Materials and Methods

Animals

All animal protocols were approved by Institutional Animal Care and Use Committee at North Carolina State University or the Marine Biological Laboratory. Female (n=27) and male (n=8) Sprague-Dawley CD IGS rats were born from timed-pregnant females purchased from Charles River (Raleigh, NC). Rats were housed with their littermates and dam until weaning at age P21. After weaning, rats were housed with same sex littermates. To ensure that rats were post-puberty, age at experimental use ranged from P70 to P112. Rooms were temperature, humidity and light-controlled (21–23°C; 12 h light:12 h darkness [34 animals]; 14h light: 10 h darkness cycle [1 animal]). Rodent chow and glass-bottle provided water were available ad libitum.

Estrous Cycle Assessment

Estrous cycle assessment was performed using a wet mount preparation as previously described (Hubscher et al., 2005). Briefly, females (P60 or older) were swabbed using potassium phosphate buffer solution (KPBS) ~10:00 am. Slides were visualized under a microscope to determine estrous cycle phase according to cell morphology as previously described (Westwood, 2008). Estrous cycle phase was confirmed via assessment of plasma concentrations of progesterone, 17β-estradiol, and testosterone in 34 of 35 animals (Table 1). At sacrifice (~10:30 am), trunk blood was collected from each subject and centrifugated within 30 minutes. Harvested plasma was stored at −20°C until assessment at the Ligand Assay and Analysis Core at the University of Virginia using commercially available ELISA kits manufactured by Calbiotech (estradiol) or IBL (progesterone, testosterone). All protocols were validated based on the recommendations of the Endocrine Society (Wierman et al., 2014). Samples were run in duplicates. Intra and inter-assay percent coefficient of variations were: estradiol: 8.3, 9.9, progesterone: 5.6, 10.2, testosterone: 5.4, 7.8, respectively. The minimum detectable plasma estradiol, progesterone, and testosterone concentrations were 3 pg/mL, 0.15 ng/mL, 10 ng/dL, respectively. The maximum detectable plasma concentrations were 300 pg/mL, 40 ng/mL, 1,600 ng/dL, respectively. Plasma estradiol levels differed across estrous cycle phase in females, as expected (Butcher et al., 1974)(Table 1). Overall, plasma hormone levels matched estrous cycle phase identification from vaginal cytology assessment, confirming the validity of this method for estrous cycle phase identification.

Table 1.

Plasma sex steroid hormone concentrations in adult rats.

Hormone	Diestrus	Proestrus	Estrus	Males	Statistics (F/KW, P)
17β-estradiol (pg/mL)	3.59 ± 0.42 (10)a *	10.01 ± 1.37 (8)b	3.10 ± 0.07 (8)a *	---	16.94, 0.0002
Progesterone (ng/mL)	37.34 ± 1.16 (10)a	22.19 ± 4.79 (8)b	28.29 ± 4.69 (8)a,b	---	4.58, 0.0212
Testosterone (ng/mL)	---	---	---	5.83 ± 1.71 (8)	---

Notes: Values are mean ± SEM. Numbers in parentheses indicate animal sample size. Superscript letters indicate statistically significant differences across groups. The presence of an asterisk (*) denotes a non-normal distribution. Acronyms: Not measured, -−−.

Electrophysiology

Acute Brain Slice Preparation

Methods for preparing brain slices for electrophysiological recordings were as previously described (Dorris et al., 2014). Rats were deeply anesthetized with isoflurane gas and killed by decapitation. Trunk blood was collected at sacrifice for serum hormone levels assessment. The brain was dissected rapidly into ice-cold, oxygenated sucrose artificial CSF (s-ACSF) containing (in mM): 75 sucrose, 1.25 NaH₂PO₄, 3 MgCl₂, 0.5 CaCl₂, 2.4 Na pyruvate, 1.3 ascorbic acid from Sigma-Aldrich, St. Louis, MO, and 75 NaCl, 25 NaHCO₃, 15 dextrose, 2 KCl from Fisher, Pittsburg, PA; osmolarity 295–305 mOsm, pH 7.2–7.4. Serial 300 μm coronal brain slices containing the caudate-putamen were prepared using a vibratome and incubated in regular ACSF containing (in mM): 126 NaCl, 26 NaHCO₃, 10 dextrose, 3 KCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, 295–305 mOsm, pH 7.2–7.4) for 30 minutes at 35°C, and at least 30 minutes at room temperature (21–23 °C). Slices were stored submerged in room temperature, oxygenated ACSF for up to 5 hours after sectioning in a large volume bath holder.

Electrophysiological recording

After resting for ≥1 hour after sectioning, slices were placed in a Zeiss Axioscope equipped with IR-DIC optics, a Dage IR-1000 video camera, and 10X and 40X lenses with optical zoom. Slices were superfused with oxygenated ACSF heated to 27±1 °C (Male: 27 ± 1 °C; Female: 27 ± 1 °C, P>0.05). Whole-cell patch-clamp recordings were made from medium spiny neurons (MSNs) in the caudate-putamen (Figure 1). Recordings were made using glass electrodes (5–12 MΩ) containing (in mM): 115 K D-gluconate, 8 NaCl, 2 EGTA, 2 MgCl₂, 2 MgATP, 0.3 NaGTP, 10 phosphocreatine from Sigma-Aldrich and 10 HEPES from Fisher, 285 mOsm, pH 7.2–7.4). Signals were amplified, filtered (2 kHz), and digitized (10 kHz) with a MultiClamp 700B amplifier attached to a Digidata 1550 system and a personal computer using pClamp 10 software. Membrane potentials were corrected for a calculated liquid junction potential of −13.5 mV (Dorris et al., 2015). Using previously described procedures, recordings were made in current clamp to assess neuronal electrophysiological properties. MSNs were identified by their medium-sized somas, the presence of a slow ramping subthreshold depolarization in response to low-magnitude positive current injections, a hyperpolarized resting potential more negative than −65 mV, inward rectification, and prominent spike afterhyperpolarization (O’Donnell & Grace, 1993; Belleau & Warren, 2000).

Figure 1.

Schematic of whole-cell patch clamped medium spiny neuron (MSN) locations in male and female rat caudate-putamen. A) Male. B) Diestrus female. C) Proestrus female. D) Estrus female. MSNs recorded from different animals are indicated by different symbols; Animal identification number is at right. Acronyms: AC, anterior commissure; CPu, caudate-putamen; CC, corpus callosum; LV, lateral ventricle; NaC, nucleus accumbens core; NaSH, nucleus accumbens shell.

In a subset of recordings, oxygenated ACSF containing the GABA_A receptor antagonist Picrotoxin (150 μM; Fisher) and the voltage-gated sodium channel blocker tetrodotoxin (TTX, 1 μm, Abcam Biochemicals) was applied to the bath to abolish action potentials and inhibitory post-synaptic current events. Once depolarizing current injection no longer elicited an action potential, MSNs were voltage-clamped at −70 mV and miniature excitatory post-synaptic current events (mEPSCs) were recorded for five minutes. This preparation detects AMPA-mediated mEPSCs and has been confirmed by our laboratory (Proano et al., 2018). Input and series resistance was monitored for changes and cells were discarded if resistance changed more than 20% or featured action potential amplitudes of less than 35 mV.

Data analysis

Basic electrophysiological properties and action potential characteristics were analyzed using pClamp 10. After break-in, the resting membrane potential was first allowed to stabilize ~1–2 minutes, as in (Mu et al., 2010). At least three series of depolarizing and hyperpolarizing current injections were applied to elicit basic neurophysiological properties (Dorris et al., 2015). Most properties measured followed the definitions of (Willett et al., 2018), which were originally derived from those of Perkel and colleagues (Farries & Perkel, 2000; 2002; Farries et al., 2005; Meitzen et al., 2009). For each neuron, measurements were made of at least three action potentials generated from minimal current injections. These measurements were then averaged to generate the reported action potential measurement for that neuron. For action potential measurements, only the first generated action potential was used. Action potential threshold was defined as the first point of sustained positive acceleration of voltage (δ²V/δt²) that was also more than three times the SD of membrane noise before the detected threshold (Baufreton et al., 2005). Rectified range input resistance, inward rectification, and percent inward rectification was calculated as described previously (Belleau & Warren, 2000). The slope of the linear range of the evoked firing rate to positive current curve (FI slope) was calculated from the first current stimulus which evoked an action potential to the first current stimulus that generated an evoked firing rate that persisted for at least two consecutive current stimuli. Input resistance in the linear, non-rectified range was calculated from the steady-state membrane potential in response to −0.02 nA hyperpolarizing pulses. The membrane time constant was calculated by fitting a single exponential curve to the membrane potential change in response to −0.02 nA hyperpolarizing pulses. Membrane capacitance was calculated using the following equation: capacitance = time constant of the membrane/input resistance. mEPSC frequency, amplitude, and decay were analyzed off-line using Mini Analysis (Synaptosoft, http://www.synaptosoft.com/MiniAnalysis/). Threshold was set at a minimum value of 5 pA, and accurate event detection was validated by visual inspection.

Statistics

Given that this experiment does not feature a balanced design (i.e., the males do not exhibit an estrous cycle), we employed a hypothesis-driven statistical analysis, similar to previous studies with comparable analysis challenges (Cao et al., 2015; Hicks et al., 2016). First, to probe for baseline sex differences, two-tailed t-tests or Mann-Whitney U tests were employed to compare male and female values independent of estrous cycle phase (Prism version 6.0; GraphPad Software, La Jolla, CA). Independent of the outcome of this first analysis, female data was then disaggregated by estrous cycle phase and compared using one-way ANOVAs or Kruskal-Wallis tests. Linear regressions were then used to further analyze select datasets. Post-hoc tests employed were Newman-Keuls or Dunn’s multiple comparison tests as appropriate. Distributions were analyzed for normality with the D’Agostino and Pearson omnibus normality test. For two-tailed t-tests and Mann-Whitney U tests, actual differences between means and medians along with 95% confidence intervals are reported to indicate magnitude and directions of changes. P values < 0.05 were considered a priori as significant. Data are presented as mean ± SEM.

Results

We recorded 122 MSNs in the caudate-putamen of adult rat male and females. To meet our primary goal of understanding the contributions of biological sex and the female estrous cycle towards modulating caudate-putamen MSN function, we first conducted analyses by sex to detect any overall sex differences present and then further analyses by estrous cycle phase within the female MSN dataset to compare between female MSNs in diestrus, proestrus, and estrus. Aggregate electrophysiological properties of all recorded MSNs and relevant statistics are presented in Tables 2 and 3.

Table 2.

Electrophysiological properties of medium spiny neurons in adult rat caudate-putamen.

Property	Male	Female	Statistics D, t/U, p		Diestrus	Proestrus	Estrus	Statistics F/KW, p
Resting Membrane Potential (mV)	−89.56 ± 0.58, (30)*	−88.86 ± 0.33, (89)*	1.072 [−0.1080, 1.754], 1051, 0.0825		−88.28 ± 0.43 (31)*	−89.89 ± 0.64 (28)	−88.50 ± 0.63 (30)*	6.527, 0.0342
Delay to First Action Potential (ms)	432.34 ± 14.46, (26)	414.21 ± 8.28, (83)*	6.125 [−47.27, 20.65], 959, 0.3978		417.67 ± 14.00 (30)	414.03 ± 15.95 (24)	410.77 ± 13.81 (29)*	0.094, 0.9217
Rheobase (nA)	0.26 ± 0.019, (31)	0.27 ± 0.01, (91)	0.009416 [−0.03501, 0.05385], 0.42, 0.6755		0.25 ± 0.01 (33)a,b,c	0.25 ± 0.02 (28)b	0.32 ± 0.02 (30)c	3.70, 0.0287
AP Threshold (mV)	−52.77 ± 1.15, (31)*	−52.37 ± 0.81, (91)*	0.8300 [−2.562, 2.980], 1382, 0.8690		−51.28 ± 1.26 (33) *,a	−57.22 ± 1.20 (28)b	−49.04 ± 1.35 (30)a	18.45, 0.0007
AP Width at Half-Peak Amplitude (ms)	1.85 ± 0.07, (31)	1.81 ± 0.03, (91)	−0.03976 [−0.1741, 0.09462], 0.5858, 0.5591		1.83 ± 0.04 (33)	1.74 ± 0.08 (28)	1.86 ± 0.04 (30)	1.19, 0.6153
AP Amplitude (mV)	68.67 ± 2.27, (31)	66.65 ± 1.27, (91)	−2.023 [−7.076, 3.031], 0.79, 0.4297		65.01 ± 1.88 (33)a,b	73.34 ± 2.57 (28)a	62.20 ± 1.72 (30)b	7.506, 0.0072
AHP Peak Amplitude (mV)	−10.36 ± 0.46, (31)*	−9.10 ± 0.23, (91)*	1.428 [0.3548, 2.036], 955.5, 0.0070		−9.40 ± 0.33 (33)	−8.72 ± 0.35 (28)	−9.11 ± 0.50 (30)*	1.941, 0.3800
AHP Time to Peak (ms)	28.89 ± 1.13, (31)	28.00 ± 0.73, (90)*	−1.353 [−3.937, 1.683], 1249, 0.3871		28.84 ± 0.96 (32)	27.12 ± 1.27 (28)	27.92 ± 1.52 (30)*	1.54, 0.6841
FI Slope (Hz/nA)	137.32 ± 7.69, (31)	151.49 ± 5.27, (91)*	12.91 [−6.688, 30.04], 1197, 0.2110		148.27 ± 7.32 (33)*	170.29 ± 12.01 (28)	137.49 ± 7.24 (30)	3.918, 0.2211
Time Constant of the Membrane (ms)	10.24 ± 1.33, (30)*	8.53 ± 0.48, (89)*	−1.300 [−2.506, 0.5146], 1137, 0.2277		8.51 ± 0.65 (33)*	10.20 ± 1.22 (27)*	6.98 ± 0.50 (29)	5.412, 0.0668
Linear Range Input Resistance (MΩ)	89.44 ± 8.43, (30)*	84.82 ± 3.45, (89)*	3.461 [−12.53, 14.45], 1318, 0.9200		87.83 ± 4.94 (32)*	81.41 ± 7.67 (28)*	84.80 ± 5.43 (29)	1.319, 0.5170
Capacitance (pF)	130.05 ± 15.68, (30)*	109.41 ± 6.12, (89)*	−12.24 [−32.22, 9.978], 1168, 0.3094		101.84 ± 9.06 (33)*	136.33 ± 12.80 (27)	92.95 ± 8.72 (29)	7.744, 0.0208
Rectified Range Input Resistance (MΩ)	78.79 ± 6.59, (30)*	78.38 ± 3.30, (89)*	3.141 [−10.48, 14.35], 1298, 0.8242		80.76 ± 4.70 (32)	71.25 ± 6.39 (28)	82.63 ± 6.13 (29)	1.098, 0.3381
Percent Inward Rectification (%)	89.47 ± 1.12, (30)*	92.34 ± 0.93, (89)	1.712 [−0.6577, 5.311], 1073, 0.1098		91.92 ± 1.18 (32)a,b,c,d	88.83 ± 1.76 (28)a,c,d	96.18 ± 1.67 (29)b,d	7.111, 0.0286
Inward Rectification (MΩ)	8.48 ± 1.35, (29)*	6.38 ± 0.95, (89)*	−1.542 [−4.476, 0.6471], 1068, 0.1029		7.07 ± 1.04 (32)*,a,b,c,d	9.95 ± 2.13 (28)a,c,d	2.17 ± 1.46 (29) *,b,d	6.943, 0.0311
Minimum Firing Rate (Hz)	2.32 ± 0.34, (31)*	2.63 ± 0.18, (91)*	1 [0.0, 1.000], 1179, 0.1596		2.43 ± 0.31 (30) *	2.63 ±0.33 (30) *	2.81 ± 0.31 (31) *	1.022, 0.5165
Maximum Firing Rate (Hz)	33.23 ± 1.72, (31)	39.14 ± 0.89, (91)	5.917 [2.314, 9.520], 3.252, 0.0015		38.60 ± 1.58 (30)a,b	36.70 ± 1.55 (30)a,c	42.03 ± 1.35 (31) b	3.286, 0.0420

Notes: Values are mean ± SEM. Numbers in parentheses indicate number of neurons recorded. Shaded values indicate statistical significance. Acronyms: Action potential, AP; Afterhyperpolarization, AHP; Frequency of evoked action potentials to injected depolarizing current, FI; D = difference between medians or means followed by the 95% confidence intervals. Superscript letters indicate statistically significant differences across groups. The presence of an asterisk (*) denotes a non-normal distribution.

Table 3.

mEPSC properties recorded from medium spiny neurons in adult rat caudate-putamen.

Property	Male	Female	Statistics D, U, p		Diestrus	Proestrus	Estrus	Statistics F/KW, p
Frequency (Hz)	2.18 ± 0.35 (15)*	2.71 ± 0.24, (38)*	0.5129 [−0.07374, 1.183], 196, 0.0798		2.89 ± 0.39 (19)*	2.84 ± 0.66 (6)	2.39 ± 0.28 (13)	0.03, 0.9872
Amplitude (pA)	12.39 ± 0.77 (15)	11.70 ± 0.41, (38)*	−0.2849 [−1.930, 1.021], 245, 0.4371		11.47 ± 0.61 (19)*	13.42 ± 1.37 (6)	11.25 ± 0.49 (13)	2.20, 0.3328
Decay (ms)	3.15 ± 0.19 (15)	3.47 ± 0.10, (38)*	0.3200 [−0.06208, 0.7635], 194, 0.0731		3.52 ± 0.13 (19)*	2.80 ± 0.37 (6)	3.69 ± 0.10 (13)	4.67, 0.0967

Notes: Values are mean ± SEM. Numbers in parentheses indicate number of neurons recorded. The presence of an asterisk (*) denotes a non-normal distribution. D = difference between medians or means followed by the 95% confidence intervals. Superscript letters indicate statistically significant differences across groups

Action potential and resting membrane potential properties differ by sex or by estrous cycle phase

Individual action potentials were elicited using positive current injections (Figure 2A). We found an overall sex difference in the afterhyperpolarization peak, with male MSNs exhibiting larger afterhyperpolarization peaks than female MSNs (Figure 2B). This finding is consistent with previous recordings in prepubertal rat caudate-putamen MSNs, which likewise observed a larger afterhyperpolarization peak in male compared to female MSNs (Dorris et al., 2015). Afterhyperpolarization peak did not differ across estrous cycle state (Figure 2B). While no other individual action potential property exhibited an overall sex difference (Table 2), further analysis of the female MSNs by estrous cycle stage revealed cycle-dependent changes across the entire spectrum of MSN electrophysiological properties.

Figure 2.

Action potential properties differ by sex or by estrous cycle stage. A) Voltage response of a male, diestrus female, proestrus female, and estrus female MSN to a single depolarizing current injection at rheobase. Threshold measurements are indicated with arrows. Resting membrane potential measurements are listed to the left of each trace. B) The action potential afterhyperpolarization peak is smaller in female MSNs relative to male MSNs, and does not differ across estrous cycle phase. C) Resting membrane potential differs by estrous cycle phase, and is hyperpolarized in proestrus female MSNs compared to diestrus female MSNs. D) Action potential rheobase differs by estrous cycle phase, and is elevated in estrus female MSNs compared to diestrus and proestrus female MSNs. E) Action potential threshold differs by estrous cycle phase, and is hyperpolarized in proestrus female MSNs compared to diestrus and estrus female MSNs. F) Action potential amplitude differs by estrous cycle phase, and is elevated in proestrus female MSNs relative to diestrus and estrus female MSNs. G) The estrous cycle-dependent change in action potential threshold negatively correlates with the estrous cycle-dependent change in action potential amplitude. These properties are significantly negatively correlated when female MSNs are analyzed independent of estrous cycle phase, as well as when diestrus, proestrus, and estrus cycle phases are assessed separately. H) The delay to the first action potential does not differ by sex or by estrous cycle stage. Acronyms: AP, action potential; AHP, afterhyperpolarization *, P<0.05; **, P<0.01; ****, P<0.0001.

Regarding resting membrane potential, female MSNs in proestrus demonstrated a hyperpolarized resting membrane potential compared to female MSNs in diestrus (Figure 2C). The rheobase, or the amount of current necessary to elicit an action potential from the MSN, was dramatically elevated for female MSNs in estrus compared to female MSNs in both diestrus and proestrus (Figure 2D). Female MSNs in proestrus demonstrated a significantly hyperpolarized action potential threshold compared to female MSNs in both diestrus and estrus (Figure 2E). Consistent with this change in threshold, the action potential amplitude was significantly larger in female MSNs in proestrus compared to female MSNs in both diestrus and estrus (Figure 2F). This change in amplitude is dependent upon changes in threshold. Supporting this interpretation, action potential threshold demonstrates a significant negative correlation with action potential amplitude (Figure 2G). This indicates that neurons with hyperpolarized thresholds exhibit action potentials with increased amplitudes. Another canonical feature of MSNs is a pronounced delay to the first action potential, reflecting the presence of the slowly inactivating A-type K current, responsible for the slow ramping subthreshold depolarization (Nisenbaum et al. 1994). No sex or estrous cycle phase differences were observed in this property (Figure 2H). Likewise, no sex or estrous cycle stage differences were detected in action potential width at half-peak (Table 2). Collectively, these data indicate MSN action potential properties are sensitive to the estrous cycle. Furthermore, when sex is analyzed independent of estrous cycle, the majority of these differences in action potential properties remain undetected.

Medium Spiny Neuron excitability differs by sex in an estrous cycle-dependent manner

Given that there were differences in rheobase and other action potential properties detected between cycle phases within females, this suggests that there may be differences in the overall excitability of MSNs at least as indicated by evoked firing rate properties (Figure 3A). To assess this possibility, we plotted the frequency of action potentials evoked by depolarizing current injection curves for individual caudate-putamen MSNs to compare the contributions of biological sex (Figure 3B) and estrous cycle phase (Figure 3C). Note that Figures 3B and 3C depict evoked action potential rate versus injected current curves up to each MSN’s maximum evoked action potential firing rate. After that point, MSN firing rate either remained constant or decreased. The maximum evoked firing rate was significantly increased in female caudate-putamen MSNs compared to male MSNs (Figure 3D). This sex difference is estrous cycle phase-dependent. Female caudate-putamen MSNs in estrus exhibit higher maximum evoked firing rates than female caudate-putamen MSNs in proestrus. To further explore the role of the estrous cycle in modulating maximum evoked firing rate and rheobase, we then analyzed whether these excitability metrics correlated with one another in female MSNs (Figure 3E). Across female MSNs regardless of estrous cycle phase, maximum firing rate and rheobase positively correlated with each other (Figure 3E, F = 10.28, R²=0.11, P=0.0019). This association was driven by MSNs recorded during proestrus (F = 11.70, R²=0.34, P=0.0023), but not diestrus (P=0.8259) or estrus (P=0.0786). This indicates that these properties are concomitant during select estrous cycle phases such as proestrus. To assess a different aspect of cellular excitability, for individual MSNs we also calculated the slope of the evoked firing rate to positive current curve (FI Slope, Figure 3F). In a previous study (Dorris et al., 2015), this was a property that was significantly increased in female compared to male prepubertal caudate-putamen rat MSNs. In adult MSNs, no sex difference in FI Slope was statistically detected, nor was this property significantly modulated across the estrous cycle in females, although a non-significant increase in females compared to males and during female proestrus is noted (Figure 3F). Similarly, no sex or estrous cycle differences were detected in minimum evoked firing rates (Table 2). Overall, these data indicate that excitability is robustly increased in females compared to males, with estrous cycle phase prominently modulating female MSN maximum evoked action potential firing rate.

Figure 3.

Action potential firing rates evoked by depolarizing current injections vary by estrous cycle phase. A) Action potential firing rates evoked by depolarizing current injections from MSNs recorded in a male, diestrus female, proestrus female, and estrus female. The number of action potentials elicited for each stimulation is listed above each trace. B) Action potential firing rates evoked by depolarizing current injections of individual MSNs differs by sex. C) Action potential firing rates evoked by depolarizing current injections of individual female MSNs differs by estrous cycle phase. D) The maximum action potential firing rate evoked by positive current stimulation differs by sex and estrous cycle phase. Maximum firing rate is greater for female MSNs compared to male MSNs, and is elevated in estrus female MSNs relative to proestrus females. E) Across female MSNs regardless of estrous cycle phase, maximum firing rate and rheobase positively correlated with each other. These properties likewise correlate during the proestrus phase, but not in the diestrus or estrus phases. F) The slopes of the evoked firing rate to positive current curve (FI Slope) in individual MSNs does not differ by sex or by estrous cycle stage. Acronyms: FI Slope: slope of the evoked action potential to depolarizing current injection curve; *, P<0.05; **, P<0.01.

Caudate-putamen medium spiny neuron input resistance and inward rectification differ across the estrous cycle in females

In response to hyperpolarizing current pulses, MSNs typically exhibit input resistance that varies strongly with membrane potential and marked time-independent inward rectification that is especially noticeable at highly negative membrane potentials (Kawaguchi et al., 1989). We assessed these properties in males and females in different phases of the estrous cycle by injecting a series of increasingly hyperpolarizing current pulses (Figure 4A). One female MSN in diestrus exhibited values over 10 standard deviations from the mean and was excluded as an outlier in analyses associated with Figures 5B–5F. Considered independent of estrous cycle phase, we detected no differences between males and females in the input resistance in both the linear and rectified ranges (Figure 4B). However, when considered in the context of estrous cycle, prominent differences seemed to emerge in the rectified but not linear ranges (Figure 4C). To further explore this possibility, we tested whether the measured input resistance differed between the linear and rectified ranges as expected from previous work in MSNs of unspecified sex. This expected decrease in input resistance was present in all groups except for female caudate-putamen MSNs in estrus (Figure 4D; P<0.0001 for diestrus and proestrus phases; P=0.1498 for estrus), which demonstrated little to no decrease. To confirm the apparent diminution of inward rectification in estrus, we assessed the total magnitude of inward rectification (Figure 4E). No sex difference was detected in this measure. However, a significant effect of estrous cycle phase was detected with female MSNs in estrus showing the least amount of inward rectification. To determine if this difference was mediated by a difference in the rectified range input resistance within each MSN, we normalized the data by calculating the percent inward rectification (percent inward rectification = rectified range input resistance/linear range input resistance × 100). Using this metric, a percent inward rectification value of 100% indicates that no inward rectification is present. As predicted by the previous measures, we detected a significant difference between female caudate-putamen MSNs in proestrus and estrus with MSNs in estrus displaying significantly less rectification (Figure 4F).

Figure 4.

Inward rectification and capacitance vary by estrous cycle phase. A) Voltage responses of a male, diestrus female, proestrus female, and estrus female MSNs to a series of hyperpolarizing current injections. B) The injected current to steady stage voltage deflection curve (IV curve) does not differ by sex when estrous phase is not considered. C) The injected current to steady stage voltage deflection curve (IV curve) differs by estrous cycle phase. D) The expected decrease in input resistance from the linear to the rectified range differs by estrous cycle phase. The magnitude of the decrease in input resistance was small or nonexistent in estrous female MSNs. E) The total magnitude of inward rectification differs by estrous cycle phase, and is decreased in estrus female MSNs relative to other phases. F) The percent inward rectification differs by estrous cycle phase, and is decreased in estrus female MSNs relative to other phases. We note that for this metric, increased inward rectification is indicated by a decreased percent inward rectification. G) The time constant of the membrane trended towards differing by estrous cycle phase, with decreased values during the estrus phase compared to other phases. H) The membrane capacitance differs by estrous cycle phase, and is elevated in proestrus female MSNs compared to estrus females. I) Linear range input resistance and the time constant of the membrane positively correlate when female MSNs are analyzed independent of estrous cycle phase, as well as during the diestrus and proestrus phases. These values do not correlate during the estrus phase. Acronyms: *, P<0.05; **, P<0.01.

Figure 5.

Excitatory synaptic properties as assessed by miniature excitatory synaptic current (mEPSC) analysis do not differ by sex or estrous cycle state. A) Representative examples of miniature excitatory post-synaptic currents (mEPSCs) from male, diestrus female, proestrus female, and estrus female MSNs. The following mEPSC properties do not differ by sex or estrous cycle phase: (B) mEPSC frequency. (C) mEPSC amplitude. (D) mEPSC decay.

To complete our analysis of the passive properties of the membrane, we calculated the time constant of the membrane and the capacitance. While we detected no significant effect of sex or estrous cycle phase on the time constant of the membrane, there was a trend toward significance regarding estrous cycle phase with values in MSNs in estrus being decreased relative to MSNs in proestrus (Figure 4G). A significant difference in the membrane capacitance between female caudate-putamen MSNs in estrus and female MSNs in proestrus was detected (Figure 4H). This change in capacitance during proestrus was driven by changes in the time constant of the membrane as significant correlations were detected between the time constant of the membrane and the input resistance for this phase (Figure 4I) (Diestrus: F = 0.19, R²=0.01, P=0.6636; Proestrus: F = 12.91, R²=0.37, P=0.0016; Estrus: F = 0.61, R²=0.02, P=0.4404; Overall: F = 8.96, R²=0.10, P=0.0036). Collectively, these data indicate that fundamental changes in passive membrane dynamics of the neuron occur over the course of the estrous cycle in female caudate-putamen MSNs.

Excitatory synaptic properties do not differ by sex or estrous cycle phase

Previous work in prepubertal caudate-putamen and nucleus accumbens shell MSNs detected no sex differences in mEPSC properties (Dorris et al., 2015; Willett et al., 2016). However, research has indicated that MSNs in the striatal region nucleus accumbens core exhibit differences in mEPSC frequency and amplitude across the estrous cycle in adult female rats (Proano et al., 2018), and that sex-specific excitatory synapse anatomy is also present (Forlano & Woolley, 2010; Wissman et al., 2011; Wissman et al., 2012; Bayless & Daniel, 2015). Thus, it is possible that mEPSC properties may likewise differ across the estrous cycle in adult caudate-putamen. To test this hypothesis, we voltage-clamped adult caudate-putamen MSNs to −70 mV and recorded mEPSCs in the presence of TTX and PTX (Figure 5A). Similar to prepubertal caudate-putamen, we detected no effect of biological sex or estrous cycle phase in the frequency (Figure 5B), amplitude (Figure 5C), or decay (Figure 5D) of mEPSCs recorded from adult caudate-putamen MSNs. This suggests that the differences observed in overall excitability are not occurring with concomitant changes in excitatory synapse properties.

Discussion

This study illustrates how adult female caudate-putamen MSN electrophysiological properties differ across the estrous cycle, leading to cycle-dependent sex differences in properties that are otherwise masked or oversimplified when only analyzed by sex. To assess MSN excitability over the estrous cycle, we analyzed a comprehensive battery of electrophysiological properties. Aspects of MSN excitability differed across the estrous cycle, but in a complex and phase-specific manner (Figure 6). We further found that these cyclical changes are specific to intrinsic excitability and do not extend to changes in excitatory synapse properties. Across all cycle phases, the relationship between caudate-putamen MSN resting membrane potential, action potential threshold, and action potential amplitude remains consistent. Though we found that MSN resting membrane potential is hyperpolarized during proestrus compared to diestrus, we found concomitant hyperpolarization of the action potential threshold and increase in the action potential amplitude during proestrus, such that the operational range of the cell has simply been shifted to a more hyperpolarized state without a net change in that aspect of excitability. Consistent with this interpretation, rheobase remained relatively constant during the diestrus and proestrus phases. However, this consistency in rheobase value was not universal across the cycle. Indeed, during estrus, MSNs require significantly more excitatory current injection to initiate an action potential than during both diestrus and proestrus. Thus, regarding action potential initiation, female caudate-putamen MSNs in estrus seem less excitable than in diestrus or proestrus. These changes in MSN resting membrane potential, threshold and rheobase implicate a number of potential targets that could be influenced by the hormones associated with the estrous cycle, including inwardly rectifying potassium currents (Jiang & North, 1991), sustained sodium and calcium currents (Kita et al., 1985; Bargas et al., 1994; Cepeda et al., 1995; Chao & Alzheimer, 1995), slowly activating potassium currents (Nisenbaum & Wilson, 1995; Shen et al., 2004), and signaling molecules such as acetylcholine and dopamine (Yasumoto et al., 2002).

Figure 6.

Schematic indicating changes in MSN electrical properties across estrous cycle phase. Acronyms: AP, action potential.

The overall interpretation that MSNs in estrus are less excitable is further supported by the behavior of MSNs in response to hyperpolarizing current injection across the cycle. During estrus, MSNs exhibit the least amount of inward rectification compared to other phases. Conversely, during proestrus, MSN inward rectification and capacitance are increased relative to both estrus and diestrus. This suggests a potential reduction in the inwardly rectifying potassium (IRK) channel conductance during the estrus phase (Mermelstein et al., 1998). It follows that this decrease in inward rectification during estrus results in a greater effect of inhibitory input onto MSNs than during proestrus and diestrus and thus a decrease in excitability if the amount of inhibitory synaptic input remains unchanged. The changes in capacitance also suggest the morphological or genetic changes could be occurring in MSNs across the estrous cycle. This question remains a future avenue of research which could greatly inform our model of cyclical changes in MSN function, especially given the presence of striatal GABAergic interneurons (Fino et al., 2018), and the known estrous-cycle and estradiol-induced changes in inhibitory neurotransmitter and synapse function in striatal and other brain regions such as the hippocampus (Staley & Scharfman, 2005; Hu et al., 2006).

Thus far, these findings seem to indicate a relatively straightforward model of cyclical modulation of female MSN excitability in which MSNs in estrus are less excitable than during the other phases. This difference in excitability, however, is specific to the properties that govern initial action potential generation and response to inhibition. A more complete picture emerges when we consider the response to strong depolarizing current injection. This is relevant to MSN physiology given that MSNs operate in multiple states including a highly-depolarizing up-state (Plenz & Kitai, 1998; Wickens & Wilson, 1998). Here we detected an overall sex difference in the maximum firing rate such that female caudate-putamen MSNs reach higher peak firing rates than males. This sex difference is primarily driven by female MSNs in estrus, which demonstrated significantly higher maximum firing rates than female MSNs in proestrus. Thus, though female MSNs in estrus require more excitation to initiate an action potential than female MSNs in proestrus and diestrus, the response of the cell to strong depolarization is significantly increased. Unlike in the nucleus accumbens core (Proano et al., 2018), we detected no changes in excitatory synapse properties across the estrous cycle, which is consistent with previous work that concentrated on dendritic spine attributes (Staffend et al., 2011; Peterson et al., 2015). This does not preclude cyclical and estradiol-induced changes in excitatory synapse activity or the activity of other inputs onto MSNs, such as those associated with the dopaminergic and cholinergic systems (Davis et al., 2005; Calipari et al., 2017).

Taken together, these findings indicate that as female caudate-putamen MSNs progress through the estrous cycle, they become more likely to initiate an action potential during diestrus than they were during estrus. This increase in excitability persists into proestrus but is accompanied by a hyperpolarization shift in resting membrane potential and action potential threshold. Then, during estrus, the resting membrane potential and threshold shift back to a more depolarized state, but the MSN requires significantly more excitation to initiate an action potential as illustrated by increased rheobase. However, MSNs in estrus are capable of producing significantly more action potentials in response to large amount of excitatory current than during both diestrus and proestrus. The multifaceted nature of estrous cycle modulation of MSN function observed here, with phase-specific changes in action potential initiation, inward rectification properties and action potential firing properties suggests the presence of multiple concurrent mechanisms. This is unsurprising given that multiple hormones fluctuate over the estrous cycle which may modulate MSN physiology.

Specifically, the estrous cycle involves fluctuations in the production of the ovarian hormones progesterone and 17β-estradiol, which are capable of crossing the blood brain barrier and acting in the brain. Decades of research have implicated estradiol in modulating striatal function (Yoest et al., 2018). Less is known regarding progesterone, although there is both recent and decades-old evidence that progesterone may also influence this brain region (Dluzen & Ramirez, 1984; Piechota et al., 2017). Focusing on estradiol, classically, estradiol acts at nuclear receptors to initiate relatively slow changes in gene transcription. However, the adult caudate-putamen does not express nuclear estrogen receptors (Almey et al., 2012), with the caveat that an exhaustive examination across all cycle phases and developmental periods has never been performed. In adult caudate-putamen, there is evidence that estradiol acts via membrane-associated estrogen receptors α, β, and GPER-1 to initiate both rapid effects on cellular function and striatal behaviors via mGluR activation, as well as triggering transcription factors such as CREB which potentially induce changes in gene expression (Mermelstein et al., 1996; Grove-Strawser et al., 2010; Almey et al., 2012; Martinez et al., 2014; Almey et al., 2015; Peterson et al., 2015; Almey et al., 2016; Martinez et al., 2016; Meitzen et al., 2018; Song et al., 2019). Expression of these membrane-associated estrogen receptors provides a possible mechanism by which the cyclical changes in estradiol over the estrous cycle may modulate MSN excitability. Though we did not measure the level of estradiol or testosterone within the caudate-putamen for this study, there is evidence that estradiol concentration reaches significantly higher levels in the female rat caudate-putamen than in blood plasma serum and varies with the estrous cycle, peaking during proestrus (Morissette et al., 1992). The striatum also expresses the enzyme aromatase, suggesting that the region may be capable of local estradiol synthesis (Kuppers & Beyer, 1998). At least one form of striatal MSN long-term potentiation has been suggested to be dependent on local estradiol action in males (Tozzi et al., 2015), but synaptic plasticity in female MSNs across the estrous cycle remains to be explored. It is possible that the changes in female caudate-putamen MSN excitability observed here are due to a multiplexed action of local estradiol synthesis, peripheral estradiol, progesterone, or the combination of all or some of these. Just as the timing of the production and release of sex hormones over the estrous cycle triggers changes in specific neural electrical attributes, it likewise triggers changes in specific animal behaviors.

These behaviors that are modulated by the hormone changes that occur over the estrous cycle all in some way facilitate successful reproduction. During the various phases of the cycle, different suites of motor and learning behaviors occur depending on the reproductive status of the rat (Calhoun, 1962; Robinson et al., 1982; Becker et al., 1987; Kent et al., 1991; Becker, 2002; Field & Pellis, 2008; Peterson et al., 2014). For example, during proestrus, female rats exhibit increased locomotor and exploratory behavior while locating and advertising to potential mates. Then, during estrus, females predominantly stay in their nest, displaying copulatory behaviors such as lordosis. During diestrus, females are no longer sexually receptive and display less exploratory and/or copulatory behaviors. Likewise, memory and learning shift in females from a hippocampal to striatal-based strategy depending on estrogen levels (Korol & Pisani, 2015). Modulation of striatal-dependent premotor and cognitive functions are required to facilitate the different requirements for successful reproduction across the cycle. Regarding how these behavioral changes relate to the estrous cycle-dependent modulation of MSN physiology, In the caudate-putamen, MSNs serve as the initial interpreter and modulator of inputs through the basal ganglia and causal relationships have been established between changes in MSN electrical function and behavioral endpoints (Kravitz et al., 2010; Kravitz et al., 2012; Cui et al., 2013; Ferguson et al., 2013; Jin et al., 2014). Thus, estrous cycle-dependent changes in MSN physiology are likely related to changes in motor and cognitive behavior. Importantly, caudate-putamen MSNs comprise at least two major pathways, which differ in neurochemistry, dopamine receptor expression, efferent targets, gene expression, functional roles, and electrophysiological properties (Gerfen et al., 1990; Cepeda et al., 2008; Gertler et al., 2008; Planert et al., 2013; Cao et al., 2018; Ho et al., 2018; Willett et al., 2019). Future research involving MSN subtype-specificity could further illuminate the functional impact of striatal hormone action. Further, convergence of afferents into the caudate-putamen are functionally organized, yielding dorsomedial and dorsolateral sub-regions, which mediate different behaviors (Fanelli et al., 2013; Ito & Doya, 2015). Here we targeted these sub-regions equally. In order to understand the relationship between sex-specific hormone modulation of neural activity and the resultant changes in behaviors, future research should further differentiate by sub-region and determine how the other neurons involved in the circuit may be concomitantly modulated. Nevertheless, estrous cycle-dependent changes in MSN physiology could ultimately yield changes in basal ganglia circuit dynamics which in turn could induce the documented estrous cycle-dependent modulation of motor and cognitive behaviors. Given that the striatum influences these altered behaviors, and that evidence indicates that estrous cycle-induced changes in MSN excitability are present in vivo, to us it is highly likely that the properties observed to differ here contribute to alterations in behavior. At this point, however, it would be speculative to link specific changes in MSN excitability and other physiological properties with specific changes in behavior.

The estrous cycle is a phenomenon specific to adulthood, but select sex differences in MSN electrophysiology are present prior to puberty. We have previously shown that MSN excitability and excitatory synaptic input differs by sex both pre- and post-puberty in region-specific, species-specific, and estrous cycle-dependent ways (Figure 7). Female rat prepubertal caudate-putamen MSNs are more excitable than male MSNs as indicated by a hyperpolarized action potential threshold, decreased action potential afterhyperpolarization, increased initial action potential firing rate, and increased slope of the evoked action potential firing rate to injected current curve (Dorris et al., 2015). This sex difference is specific to the caudate-putamen, as MSNs in rat nucleus accumbens core and shell do not exhibit sex differences in cellular excitability during the prepubertal period (Cao et al., 2016; Willett et al., 2016). Similarly, no sex differences are detected in excitatory synapse properties in the caudate-putamen and nucleus accumbens shell of prepubertal rats (Dorris et al., 2015; Willett et al., 2016). Conversely, mEPSC frequency is dramatically increased in prepubertal female compared to male nucleus accumbens core MSNs and this difference is sensitive to perinatal estradiol exposure (Cao et al., 2016). The striatal regions are altered during puberty and some sex differences do not emerge until adulthood (Andersen et al., 1997; Andersen et al., 2002; Ghahramani et al., 2014; Staffend et al., 2014; Kopec et al., 2018). In adult female rats, Proaño and colleagues recently demonstrated estrous cycle-dependent differences in nucleus accumbens core MSN excitability and excitatory synapse properties such as mEPSC frequency and amplitude (Proano et al., 2018). Here, we detected estrous cycle-dependent differences in caudate-putamen MSN excitability without changes in excitatory synapse properties. Further, the ways in which MSN excitability is modulated in the caudate putamen differ from the nucleus accumbens core. Given these region-specific neurodevelopmental differences in MSN physiology, future research should investigate for potential post-pubertal changes in nucleus accumbens shell MSN excitability and excitatory synapse properties. Additionally, these sex differences are species-specific as the prepubertal sex differences detected in rat caudate-putamen and nucleus accumbens core MSN are different from or absent in mice assessed during the same general developmental period (Cao et al., 2018; Willett et al., 2019). This foundational research into a single neuron type in varying regional and neuroendocrine contexts establishes the immense malleability of cellular function and the importance of considering not just animal sex, but age and hormone status when gathering and interpreting physiological data. This is illustrated by our chosen analysis paradigm, in which baseline sex differences were first compared independent of estrous cycle phase. Importantly, only one property out of the eight that differed by estrous cycle phase was also found to differ by sex when estrous cycle phase was not considered. While we are encouraged that increasing numbers of neuroscience studies are assessing females (Beery & Zucker, 2011; Shansky & Woolley, 2016; Will et al., 2017), studies that do not in some way disaggregate estrous cycle phases, whether similar to the approach employed here, a simultaneous multi-factorial design, or other statistical paradigm, risk missing key variables influencing neural physiology. Nevertheless, we acknowledge that not all variables can be addressed in a single study and there are multiple factors which can influence neuron function that were not assessed in this study. Overall, this body of research demonstrates the immense heterogeneity and plasticity of fundamental cellular properties in a single neuron type, the medium spiny neuron.

Figure 7.

Developmental of regional sex and estrous cycle differences in rat MSN electrophysiological properties. ↺ symbol indicates estrous cycle-dependent sex differences were detected.

Abbreviations

ACSF

artificial cerebrospinal fluid

Estradiol

17β-estradiol

KPBS

potassium phosphate buffer solution

mEPSC

miniature excitatory post-synaptic current

MSN

medium spiny neuron

s-ACSF

sucrose artificial cerebrospinal fluid

TTX

tetrodotoxin