Sexually dimorphic characteristics of dopamine D1 receptor-expressing neurons within the shell of the nucleus accumbens of adolescent mice

Background: Adolescence, a developmental stage, is characterized by psychosocial and biological changes. The nucleus accumbens (NAc), a striatal brain region composed of the core (NAcC) and shell (NAcSh), has been linked to risk-taking behavior and implicated in reward seeking and evaluation. Most neurons in the NAc are medium spiny neurons (MSNs) that express dopamine D1 receptors (D1R+) and/or dopamine D2 receptors (D2R+). Changes in dopaminergic and glutamatergic systems occur during adolescence and converge in the NAc. While there are previous investigations into sex differences in membrane excitability and synaptic glutamate transmission in both subdivisions of the NAc, to our knowledge, none have specified NAcSh D1R+MSNs from mice during mid-adolescence. Methods: Sagittal brain slices containing the NAc were prepared from B6.Cg-Tg(Drd1a-tdTomato)6Calak/J mice of both sexes from postnatal days 35–47. Stained smears were made from vaginal samples from female mice to identify the stage of Estrous at death. Whole-cell electrophysiology recordings were collected from NAcSh D1R+MSNs in the form of membrane-voltage responses to current injections and spontaneous excitatory postsynaptic currents (sEPSCs). Results: The action potential duration was longer in males than infemales. Additionally, the frequency of sEPSCs was higher in females, and the mean event amplitude was smaller than that in males. We found no evidence of the observed sex differences being driven by the stage of the Estrous cycle and no physiological parameter significantly varied with respect to the Estrous cycle. Conclusions: Taken together, our results indicate that NAcSh D1R+MSNs exhibit sex differences during mid-adolescence that are independent of the stage of Estrous, in both AP waveform and glutamate transmission, possibly due to changes in voltage-gated potassium channels and α-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors, respectively.


Introduction
Adolescence is a stage of development characterized by psychosocial and biological changes that can contribute to an increased propensity to take risks.(1,2) This tendency toward risk-taking, or choosing an action for which the outcome is more variable, is greater during puberty than childhood and increases across adolescence.(3,4) During development, risk-taking plays an important role in one's ability to de ne oneself and learn new skills, but it can also lead to reckless behavior.(1,5) Across the span of adolescence, developmental alterations in the brain are widespread, multifaceted, and work to shape the childhood brain into that of adulthood.
One brain structure that is heavily modi ed during adolescence and linked to risk-taking behavior is the nucleus accumbens (NAc), found within the ventral striatum.Implicated in the seeking and evaluation of reward,(6, 7) the NAc has two subregions: the shell (NAcSh) and the core (NAcC).Both subregions contain dopamine (DA) receptor and glutamate receptor-expressing, -aminobutyric acid-producing (GABAergic) medium spiny neurons (MSNs).NAcSh MSNs project to brain structures mediating decision making, rewarding properties of substances, and reinforcing properties of novelty, and NAcC MSNs innervate structures associated with impulsive choices, spatial learning, and responses to motivational stimuli.(8-10)MSNs have been classi ed by the subtype of DA receptor that they express: dopamine D1 receptor-expressing (D1R+) or dopamine D2 receptor-expressing (D2R+) MSNs.Both subtypes receive synaptic excitation via glutamatergic inputs from several forebrain structures, and dopaminergic (DAergic) inputs from the midbrain serve to modulate MSN membrane properties and excitability.(9,11) During adolescence, drastic changes in both the DAergic and glutamatergic systems occur, (12,13) some of which appear to be sex speci c.Striatal increases in DA concentration, transporters, receptor binding, and receptor expression have been documented as occurring during adolescence.(1,(14)(15)(16)(17) Overall, the ratio of D1R + to D2R + neurons in the ventral striatum increases during adolescence, with female mice exhibiting a heightened ratio compared to males.(18)Male D1R expression and binding peaks during adolescence, whereas female D1R expression appears to peak prior to adolescence, and although striatal D1R pruning occurs in both sexes, it occurs at different ages and via different mechanisms in males and females.(19,20) Regarding glutamate, both the frequency and amplitude of spontaneous excitatory postsynaptic currents in NAc MSNs of male rats were shown to decline during adolescence.(21) Therefore, it is likely that both DAergic and glutamatergic actions in the NAc play a role in the transition from childhood to adulthood.
Puberty, or attaining sexual maturation, occurs during adolescence, is initiated by the brain and is accompanied by increases in gonadal hormones.(1) The terms adolescence and puberty are not synonymous, and the age at which each occurs varies by species, strain, sex, and individual.In rodents, adolescence is considered to occur between postnatal day (PND) 30 and PND 49. (22) In female mice, puberty and rst estrus occur between 10-20 days after vaginal opening on PND 26, depending upon the strain.(23,24) For male mice, puberty is marked by motile sperm, which occurs between PND 40 and PND 55. (25,26) Although previous bodies of work have investigated whether MSNs in the NAc exhibit sex-related differences during adolescence and possible effects of the Estrous cycle, (27)(28)(29) none of these studies addressed pubertal age directly or speci cally evaluated D1R + MSNs in the NAcSh of mice.Considering the evidence for DAergic and glutamatergic changes in the striatum during adolescence, that DA modulates MSN excitability, and that glutamate functions as a major excitatory neurotransmitter in the brain, we examined membrane excitability and synaptic glutamate transmission in NAcSh D1R + MSNs from mid-adolescent mice of pubertal age, gonads intact, to bridge this gap in knowledge.

Subjects
All whole-cell electrophysiology recordings were conducted in brain slices prepared from male and female bacterial arti cial chromosome transgenic B6.Cg-Tg(Drd1a-tdTomato)6Calak/J mice (developed by Ade and colleagues (30) (RRID:IMSR_JAX:016204)) from a colony maintained in our laboratory.Animals were group-housed on a 12:12 reverse light cycle (ZT0 = 21:30) in standard mouse cages containing wood chip bedding (Sani-Chips; PJ Murphy).Animals were provided with enrichment in the form of compressed cotton ber squares (Nestlets; Ancare).Food (Prolab®5LL2 RMH 1800; LabDiet) and water were supplied ad libitum.On the day of the experiment, the mice ranged in age from PND 35-PND 47, representing midadolescence; (31) the mean age of males was PND 39, and the mean age of females was PND 39 (t(80) = 0.3195, p = 0.7502) a (Fig. 1a).
All animal procedures were performed in accordance with the University of Texas at Austin's institutional animal care and use committee's regulations.

Timing of the Estrous Cycle
Vaginal samples were collected from all female mice immediately following decapitation.A micropipette tip was inserted 1-2 mm into the vaginal opening and 10 µL of 0.9% saline was ushed into the vagina three times.The resulting sample was then applied to a glass slide and a smear was made.Each smear was allowed to dry fully before staining with Wright-Giemsa stain.Under 20X magni cation, each smear was analyzed for the presence of nucleated epithelial cells, anucleated epithelial cells, and leukocytes and was classi ed as representing proestrus, estrus, metestrus, or diestrus as described by Cora and colleagues.(32) Data Acquisition Whole-cell recordings were collected from D1R + MSNs located in the NAcSh (Fig. 1b).Cells were identi ed as D1R + by the presence of epi uorescent illumination of tdTomato using the MRK200 Modular Imaging system (Siskiyou Corporation).Data was acquired utilizing a CV203BU headstage mounted on a vibration isolation table and an Axopatch 200B ampli er, with 1 kHz ltering.Data was digitized at 5 kHz through a Digidata 1440A interface board using Clampex 10.3 (all products by Molecular Devices, Sunnyvale, CA, United States).Immediately after obtaining whole-cell con guration, cells were selected for further experimentation by exhibiting a series resistance of less than 33 MΩ and having a resting membrane potential of less than or equal to -60 mV.Any electrophysiology recording during which series resistance changed by more than 20% or exceeded 33 MΩ was excluded from statistical analysis.Membrane voltage responses were measured by recording 20 sweeps 750 ms in duration that included in order: 50 ms of no current application, 150 ms of -20 pA, 150 ms of no current application, 300 ms of either a hyperpolarizing or depolarizing current, and 100 ms of no current application.Hyperpolarizing or depolarizing current steps increased at a rate of 50 pA from − 400 pA to 550 pA.The resting membrane potential was de ned as the average voltage (mV) during the initial 50 ms of the 0 pA sweep.The steady state of the voltage response to the − 20 pA step was used to calculate the input resistance.Rheobase was de ned as the amplitude of the current step that elicited the rst action potential (AP) for each cell.AP waveforms were analyzed using the rst AP in the rheobase step, unless the AP red too close to the end of the step to measure AHPs; in these cases, the rst AP of the next step was used.The AP threshold was de ned as the point at which dV/dt exceeded 10 mV/ms for the rst AP.AP amplitude was calculated by subtracting the AP threshold from the AP peak voltage.AP half-width was de ned as the duration between the AP threshold and the AP half-amplitude.AP afterhyperpolarization potential (AHP) amplitudes were de ned as the difference between the threshold voltage and the most negative voltage within 5 ms of threshold, and the voltages at 10 and 15 ms after the AP threshold, for fast (fAHP), medium (mAHP) and slow (sAHP), respectively.(33,34) The sweep with the maximum number of APs was used to determine the spike frequency adaptation ratio (SFA) and to determine the maximum peak-to-peak frequency using the shortest interval between two APs.SFA was calculated by dividing the interval between the rst two APs of the sweep by the interval between the last two APs of the sweep.To monitor spontaneous excitatory postsynaptic currents (sEPSCs), the membrane potential was clamped at -80 mV.Raw data analysis was performed in Clamp t 10.6 (Molecular Devices).Data was acquired blind to the stage of the Estrous cycle.Data analysis took place after all data was collected and was performed blind to sex and stage of Estrous.The sEPSC frequency and average amplitude were determined over a 1-3-minute period by utilizing the Clamp t template search feature and rejecting events smaller than 5 pA.

Statistics
Statistical analyses were performed in GraphPad Prism 9.3.The current step-evoked action potential number was analyzed by two-way ANOVA, with sex or stage of Estrous as the between-group factor and the current step as the repeated measure.When comparing sex, variables were analyzed using unpaired Student's t tests; Welch's correction was applied when homogeneity of variance was violated.When comparing the stages of Estrous, variables were analyzed using a one-way ANOVA or Kruskal-Wallis test when data were both nonnormally distributed and in violation of homogeneity of variance.Depiction of variance on gures was speci c to allow for better visualization.Table 1 shows details of the statistical analysis.
We then wished to investigate whether the sex differences we observed could be attributed to a stage or stages of the Estrous cycle and grouped cells from female mice into proestrus, estrus, metestrus, or diestrus for comparison.We observed no signi cant difference between stages of Estrous for any measure of D1R + MSN membrane properties nor cellular excitability, including: resting membrane  4).Additionally, spontaneous glutamatergic transmission did not differ by stage of Estrous, as no signi cant difference was observed for the frequency of spontaneous excitatory events (F(3, 58) = [2.064],p = 0.1149) ab or for average event amplitude (F(3, 58) = [1.177],p = 0.3262) ac (Fig. 5).

Discussion
Our results indicate that D1R + MSNs in the NAcSh differ by sex in action potential (AP) waveform and spontaneous glutamatergic transmission.In summary, we found AP duration to be longer in males and that females exhibited a lower amplitude but increased frequency of sEPSCs.There was no evidence that any one stage of the Estrous cycle drove the observed sex differences or a lack thereof.To our knowledge, this is the rst body of work to focus on D1R + MSNs in the NAcSh during mid-adolescence in pubertal aged mice.
Here, we observed a longer AP duration in D1R + MSNs from male mice relative to females.The width of the mammalian action potential waveform is in uenced by the activation of both voltage-gated sodium (Na v ) and potassium (K v ) channels.(35) While blocking Na v channels has previously been shown to lengthen AP duration in MSNs in the NAc of adolescent rats, this manipulation also resulted in a reduction in AP amplitude.(36)As we found no evidence of sex differences in AP amplitude, our results suggest that rather than Na v channels underlying the observed sex difference, K v channels may be responsible.Activation of K v channels limits cellular excitability by repolarizing the membrane after Na v channels close.(37) Although there are many subunits of K v channels expressed in the striatum, recent ndings from Otuyemi et al. (38) indicate that, at least in the dorsal striatum, D1R + MSNs from adult mice of both sexes express K v 2.1 and K v 4.2 channels.The localization of K v 2.1 (distributed across the soma and proximal dendrites) suggests that these channels may have contributed to our observed sex difference in AP waveform, rather than K v 4.2 channels (which are on distal dendrites).K v 2.1 channels may be found in non-conducting clusters or in conducting non-clusters, and in response to glutamate, clustered K v 2.1 channels can disperse across the cell surface.(38-41)Therefore, it is plausible that during mid-adolescence, the number of and/or clustering patterns of K v 2.1 changes over murine ontogeny, possibly in response to glutamatergic transmission, and that the ontogeny of K v 2.1 is impacted by sex but not by the stage of the Estrous cycle.Further evidence supporting this notion can be found in the work of Brundage and colleagues (42), who also observed evidence for sex differences in the function of K v channels in the striatum of mice from PND 30 onward.
Spontaneous excitatory postsynaptic currents (sEPSCs) differed in both frequency and amplitude in a sex-dependent manner.Here, we observed D1R + MSNs in the NAcSh of female mice to have a decreased event amplitude and an increased event frequency when compared to the same cell type in males.As cells were held at -80 mV during these electrophysiology recordings, N-methyl-D-aspartate (NMDA)-type ionotropic glutamate receptors can be eliminated as strong contributors to this sex difference due to the magnesium block at this voltage.(43) Kainate receptors (KAR) GluR6, GluR7 and KA2 are expressed in the striatum of mice and rats, (44)(45)(46)) and as we did not pharmacologically isolate AMPARs here, we cannot exclude KAR involvement in the sex differences observed for sEPSC frequency or amplitude.However, when considering that the majority of KARs are expressed outside of the postsynaptic density,(47) their involvement as a major contributor to sex differences in EPSCs appears unlikely, although to our knowledge, KAR distribution has not yet been investigated during mid-adolescence in both sexes.Thus, αhydroxy-5-methyl-4-isoxazolepropionic acid type ionotropic glutamate receptors (AMPARs) are likely the predominant mediators of the observed sex differences in excitatory synaptic transmission.
Functional AMPARs are present on mature synapses, function in basal neurotransmission, and have been shown in other brain regions to have a high likelihood of being present on large dendritic spines.(48-52)Previous work by Forlano and Woolley (53) suggests that female MSNs in the NAc exhibit a greater dendritic spine density and a greater number of large-headed spines than males.Furthermore, during adolescence, synaptic pruning of D1R + and D2R + MSNs has been observed to be greater in males.(1)Thus, it is plausible that the increased SEPSC frequency observed in females is a re ection of having a greater number of functional synapses than males.In addition to synapse number, overall network activity in uences sEPSC frequency.Furthermore, differences in frequency can be mediated by both preand postsynaptic mechanisms, such as altered presynaptic release probability or insertion of AMPARs into previously "silent" synapses, respectively.(54)The latter phenomenon, through which postsynaptic AMPAR insertion results in increased EPSC event frequency, is a well-documented developmental event.
(54) This again points toward differences in the number of functional synapses as a plausible explanation for the increased event frequency in females.
Our ndings do not elucidate what speci c alteration or combination of changes in AMPARs contributed to the sex difference in amplitude observed here.Changes in the number of AMPARs expressed on the cell surface, incorporation of GluA2 subunits, alternative splicing of the ip-op module, and editing at the R/G site can in uence the amplitude of postsynaptic currents by impacting desensitization, recovery from desensitization, and single channel conductance.(55)(56)(57)(58)(59)(60)(61)(62) There is also evidence that the expression, editing, and alternative splicing of AMPARs changes over ontogeny in both mice and rats.(59,63, 64) For example, editing at the Q/R site, which reduces single channel conductance, is completed prior to parturition in mice.(65,66) Thus, our ndings highlight potential sex differences in AMPAR ontogeny.
To our knowledge, only Willett and colleagues (27) have reported on excitatory synaptic transmission and membrane properties of NAcSh MSNs from both sexes, nding no evidence of signi cant sex differences.Their work was conducted in rats at PND 21 in MSNs not identi ed by subtype, and the recorded neurons were in an area of the NAcSh that was more dorsal to that in our study.Thus, one interpretation of our nding of signi cant sex differences in the NAcSh during PND 35-47, while Willett and colleagues did not nd any at PND 21, could be that sex differences in glutamate transmission and action potential halfwidth emerge between pre-and mid-adolescence.On the other hand, the discrepancy in ndings might also be a result of important methodological differences.Therefore, to better understand exactly when and where sex differences emerge, future studies should target speci c populations of MSNs at multiple developmental stages.

Figures
Figures

Figure 1 Age
Figure 1

Table Table 1
4. Petralia RS, Esteban JA, Wang YX, Partridge JG, Zhao HM, Wenthold RJ, et al.Selective acquisition of AMPA receptors over postnatal development suggests a molecular basis for silent synapses.Nat is available in the Supplementary Files section.