Sexually dimorphic neuronal responses to social isolation
Survival threats trigger a repertoire of behavioral and endocrine adjustments ( Cannon , 1932 ) . The endocrine changes rely on the immediate engagement of the hypothalamic pituitary adrenal ( HPA ) axis which is controlled by corticotropin releasing hormone ( CRH ) neurons in the paraventricular nucleus of the hypothalamus ( PVN ) . Although it is essential for survival , the dysregulation of the stress response is implicated in the emergence of numerous neuropsychiatric diseases ( Tost et al . , 2015 ) . Interestingly , many of these psychopathologies show sex-specific differences which may be a consequence of sexual dimorphism of the neuroendocrine response to stress ( Bale and Epperson , 2015 ) . This sexual dimorphism may result in different response sensitivity to the same stressor and/or differences in which stimuli males and females perceive as stressful . Human studies demonstrating that young girls exhibit greater corticosteroid ( CORT ) stress reactivity to social stress tests than boys ( Gunnar et al . , 2009; de Veld et al . , 2012 ) suggest , for example , that young females are more responsive to changes in social situations . Consistent with this idea , are demonstrations that , in comparison to males , females take greater advantage of social support and group dynamics to manage stress ( Taylor et al . , 2000 ) . Here we hypothesized that disrupting the social network would elicit neurobiological changes preferentially in female mice .
To test this idea , mice were housed in same-sex groups ( 3–5 animals per group ) from weaning ( p21 ) until the day of the experiment ( p22–35 ) . This developmental window allows us to interrogate the contributions of sex differences independent of the effects of circulating gonadal hormones ( Nelson et al . , 1990 ) . Mice were either in same-sex groups , pairs , or isolated from littermates for sixteen to eighteen hours . We then prepared hypothalamic brain slices and examined synaptic and intrinsic properties of identified CRH neurons in the PVN in acute brain slices ( Wamsteeker Cusulin et al . , 2013b ) ( Figure 1a ) . Activation of these cells is obligatory for launching the neuroendocrine response to stress ( Denver , 2009 ) . 10 . 7554/eLife . 18726 . 003Figure 1 . Social isolation alters intrinsic properties of CRH neurons in female , not male mice . ( a ) Experimental timeline ( top ) , schematic ( middle ) showing coronal brain section of PVN ( red ) with a fluorescent image of tdTomato immunofluorescence ( bottom ) . Panels on right show DIC image with electrode ( top ) , tdTomato cells ( middle ) and recorded neuron ( bottom ) ( b ) Summary bar graphs indicate no difference in FSL for single and group-housed males ( left , one-way ANOVA , p=0 . 9 ) . FSL in single females is significantly longer than group-housed females ( one-way ANOVA , p<0 . 0001 ) . FSL in group-housed females and males are not different ( p>0 . 05 ) . ( c ) Traces show responses to a single +80 pA depolarizing step ( holding potential = −102 mV ) in group ( dark blue ) and single-housed ( light blue ) males . FSL calculated from the start of the depolarizing pulse ( dotted line ) to the first spike is plotted for all cells in ( c ) . The relative frequency distributions are overlaid by the relative cumulative distributions . ( d ) Traces show responses to the same depolarizing step protocol as in ( c ) but in group ( dark orange ) and single-housed ( light orange ) females . The relative frequency distributions are overlaid by the relative cumulative distributions . DOI: http://dx . doi . org/10 . 7554/eLife . 18726 . 00310 . 7554/eLife . 18726 . 004Figure 1—figure supplement 1 . No sex differences in basal properties of CRH neurons . ( a ) Left: Input resistance of individual cells from isolated males and females ( male: 797 ± 32 MΩ , n = 157 , vs female: 868 ± 23 MΩ , n = 273; unpaired t-test , p=0 . 06 ) . Right: Spike thresholds of individual cells from isolated males and females ( male: −51 . 5 ± 1 . 0 mV , n = 63 vs female: −51 . 3 ± 0 . 7 mV , n = 99; unpaired t-test , p=0 . 86 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18726 . 00410 . 7554/eLife . 18726 . 005Figure 1—figure supplement 2 . No sex differences in excitatory post-synaptic currents . ( a ) Representative traces of spontaneous synaptic activity from individual cells from single-housed males and females with bath application of picrotoxin ( 100 µM ) . ( b ) Summarized data of cumulative fraction of amplitude ( pA ) and inter-event intervals . There were no significant differences in either the spontaneous amplitude ( males: 34 . 14 ± 1 . 30pA , n = 12; females: 33 . 10 ± 1 . 19pA , n = 45 , p>0 . 05 ) or frequency ( males: 2 . 99 ± 0 . 61 Hz , n = 12; females: 2 . 50 ± 0 . 25 Hz , n = 45 , p>0 . 05 ) . ( c ) Peak scaled sample trace of a paired-pulse recording from a male ( blue ) and a female ( orange ) . Right: Summary data of PPRs recorded from males and females . There was no significant difference in the paired pulse ratio ( PPR ) of EPSCs ( males: 0 . 72 ± 0 . 05 , n = 9; females: 0 . 80 ± 0 . 04 , n = 32 , p>0 . 05 ) . Scale bars are 50 pA/10 ms . DOI: http://dx . doi . org/10 . 7554/eLife . 18726 . 00510 . 7554/eLife . 18726 . 006Figure 1—figure supplement 3 . No sex differences in inhibitory post-synaptic currents . ( a ) Representative traces of spontaneous synaptic activity from individual cells from males and females with bath application of DNQX ( 10 µM ) . ( b ) Summarized data of cumulative fraction of amplitude ( pA ) and inter-event intervals . There were no differences in sIPSC amplitude ( males: 27 . 61 ± 0 . 89 pA , n = 43; females: 26 . 77 ± 0 . 76 pA , n = 66 , p>0 . 05 ) or frequency ( males: 2 . 25 ± 0 . 27 Hz , n = 43; females: 1 . 85 ± 0 . 14 Hz , n = 66 , p>0 . 05 ) . ( c ) Peak scaled sample trace of a paired-pulse recording from a male ( blue ) and a female ( orange ) . Right: Summary data of PPRs recorded from males and females . There was no significant difference in the PPR of IPSCs ( males: 0 . 65 ± 0 . 02 , n = 29; females: 0 . 70 ± 0 . 02 , n = 57 , p>0 . 05 . Scale bars are 50 pA/10 ms . DOI: http://dx . doi . org/10 . 7554/eLife . 18726 . 006 The cellular heterogeneity in the PVN has necessitated various approaches to distinguish cell types . One of the most widely used has been to identify cells based on their voltage responses to a protocol of current steps . This reveals an ‘electrical fingerprint’ that has been used to distinguish among magnocellular ( oxytocin , vasopressin ) ( Tasker and Dudek , 1991 ) and parvocellular ( CRH , TRH , somatostatin ) neurosecretory cells ( Hoffman et al . , 1991 ) and pre-autonomic neurons . Reports of changes in the intrinsic properties of neurons following experimental manipulations ex vivo ( Shah et al . , 2010; O'Leary et al . , 2014; Kourrich et al . , 2015 ) , however , raise the possibility that electrical fingerprints are dynamic and potentially unreliable as a classification tool . We have recently described a transgenic reporter mouse in which CRH neurons can be identified and targeted directly for electrophysiological recording ( Wamsteeker Cusulin et al . , 2013b ) . In male mice , the electrical fingerprint of CRH cells was consistent with previous reports on parvocellular neurosecretory cells ( Wamsteeker Cusulin et al . , 2013b ) . These cells had a linear current voltage relationship and , unlike the magnocellular neurosecretory cells , did not have a long delay to first spike ( first spike latency ( FSL ) ) when depolarized from hyperpolarizing membrane potentials . We first examined FSL of CRH neurons from group-housed vs isolated mice . FSL can play a key role in integrating synaptic events and influence probability of firing ( Molineux et al . , 2005 ) and changes in the underlying currents can tune neuronal frequency ( Ellis et al . , 2007 ) . Social isolation had no effect on FSL in males ( malegroup: 48 . 7 ± 2 . 1 ms , n = 53 vs malesingle: 46 . 1 ± 1 . 8 ms , n = 76 , p=0 . 9; Figure 1b , c ) . By contrast , FSL in isolated females was significantly longer than FSL in group-housed females ( femalegroup: 45 . 1 ± 1 . 9 ms , n = 42; femalesingle: 65 . 6 ± 1 . 9 , n = 177 , p<0 . 0001; Figure 1b , d ) . We then conducted experiments in which female mice were housed in pairs prior to electrophysiology experiments . This manipulation revealed an intermediate phenotype , with pair-housed females exhibiting FSLs that were longer than those observed in group housed female mice , but shorter than those observed in single-housed female mice ( femalepaired: 56 . 5 ± 2 . 0 ms , n = 105 , p=0 . 0029 vs femalesingle and p=0 . 0017 vs femalegroup , F4 , 471 = 10 . 6 ) . There was no difference in the input resistance of isolated males vs isolated females ( malesingle: 796 ± 31 MΩ , n = 157 , vs femalesingle: 869 ± 23 MΩ , n = 273; unpaired t-test , p=0 . 063; Figure 1—figure supplement 1 ) or spike threshold ( malesingle: −51 . 5 ± 1 . 0 mV , n = 63 vs femalesingle: −51 . 3 ± 0 . 7 mV , n = 99; unpaired t-test , p=0 . 86; Figure 1—figure supplement 1 ) . We also examined properties of glutamate and GABA synapses on CRH neurons in hypothalamic slices from single-housed females and males . We observed no differences in basal glutamate ( Figure 1—figure supplement 2 ) or GABA ( Figure 1—figure supplement 3 ) synaptic transmission . These observations indicate that social isolation alters the intrinsic properties of CRH neurons in female , but not age-matched male mice . Next , we hypothesized that female mice , but not male mice , may interpret social isolation as a stress . If true , we reasoned that subjecting single-housed male mice to an acute swim stress should also increase FSL . In females , however , the prior isolation should occlude the effects of an acute stress . To test this idea , single -housed male and female mice were subjected to swim stress for 20 min and intrinsic properties of CRH neurons were assessed ( Figure 2a ) . Following swim stress , FSL in male mice ( maleswim ) was 64 . 0 ± 4 . 6 ms , n = 37 , Figure 2b , c; this is significantly longer than FSL in the single males reported above ( maleswim vs malesingle , 1 way ANOVA , p=0 . 0004 ) . FSL in single female mice subjected to swim stress was 65 . 8 ± 4 . 9 ms , n = 32 , Figure 2b , c; this is not different from FSL in female mice subjected to social isolation ( femaleswim vs femalesingle , p=0 . 67 ) . Finally , FSL in single males subjected to swim was not different from FSL in single females subjected to swim ( p=0 . 9 , Figure 2b , c ) . This suggests that either increases in FSL following social isolation occlude further effects of stress on CRH neurons or that females do not respond to swim stress . To test the effects of swim stress independent of any potential effects of social isolation , we conducted experiments in which group-housed females and males were subjected to swim stress . Following swim , FSL in group-housed males ( malegroup swim: 58 . 8 ± 2 . 8 ms , n = 23 , Figure 2d ) was significantly longer than FSL in naïve , group -housed males ( p<0 . 001 ) . FSL of group-housed males subjected to swim was not different than FSL in females subjected to swim ( femalegroup swim:58 . 7 ± 1 . 9 ms , n = 27 , p>0 . 05 vs malegroup , Figure 2d ) . Similarly , CRH neurons in group-housed females subjected to swim had a significantly longer FSL than CRH neurons in naive , group-housed females ( p=0 . 0001 ) . These observations demonstrate that both females and males show equivalent sensitivity to an acute physical stress ( i . e . forced swim ) . Additionally , a prior social isolation occludes the effects of subsequent physical stress on FSL in females , but has no effect on FSL in males . 10 . 7554/eLife . 18726 . 007Figure 2 . Females and males show equivalent sensitivity to an acute physical stress . ( a ) Experimental timeline . ( b ) Neuronal responses to +80 pA depolarizing pulse in single-housed males and females . ( c ) Summary data show that following swim stress , FSL in single males and single females is not different ( p>0 . 05 ) . FSL in single males subjected to swim is significantly longer than FSL in naive single males ( p<0 . 0001 ) . FSL in single females subjected to swim is not different than single females ( p>0 . 05 ) . ( d ) Summary data show that FSL in group-housed males and females is not different ( p>0 . 05 ) . Group-housed males subjected to swim have longer FSL than group-housed , naive males ( p<0 . 001 ) and group-housed females subjected to swim have longer FSL than group-housed , naive females ( p=0 . 0001 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18726 . 007 Since stress increases CORT , we examined the relationship between circulating CORT and FSL . In order to assess CORT during a defined temporal window , we subjected single male mice to swim stress and obtained samples thirty minutes after the protocol . We noted a positive correlation between plasma CORT and FSL ( r2 = 0 . 3 , p=0 . 024 , n = 17 , Figure 3a ) . CORT , however , is one of many signaling molecules that is altered in response to stress . To determine whether CORT is necessary for increases in FSL following social isolation , we conducted experiments in which female mice were given access to drinking water containing the CORT-synthesis inhibitor , metyrapone prior to , and during , isolation ( Figure 3b ) . Metyrapone treatment blocked the effects of social isolation on FSL in female mice ( femalesingle metyrapone = 47 . 2 ± 2 . 0 ms , n = 24 , p=0 . 001 vs femalesingle , Figure 3c ) . Next , to determine whether CORT is necessary for increases in FSL following swim stress , we administered metyrapone prior to swim stress in single males ( Figure 3d ) . This eliminated the stress-induced increase in FSL in males ( malemetyrapone = 50 . 2 ± 2 . 4 ms , n = 35 , vs malevehicle = 66 . 7 ± 2 . 4 , n = 23 , p=0 . 007 , Figure 3e ) . These observations demonstrate that CORT is necessary for stress-induced changes in FSL in both males and females . In order to determine whether CORT is sufficient for increasing FSL , we incubated slices from either single-housed males or group-housed females with 100 nM CORT for 1 hr prior to electrophysiological assessment . This incubation time is sufficient to mimic CORT-dependent changes in synaptic metaplasticity observed after acute stress ( Wamsteeker Cusulin et al . , 2013a ) . CORT-incubation increased FSL in slices from single-housed males ( 59 . 2 ± 4 . 1 ms , n = 36 , p<0 . 021 vs malesingle ) and in slices from group-housed females ( 64 . 1 ± 4 . 8 ms , n = 32 , p<0 . 0001 vs femalegroup , Figure 3f ) . Collectively , these observations demonstrate that CORT is both necessary ( during stress ) and sufficient ( in the absence of stress ) to increase FSL in male and female mice . 10 . 7554/eLife . 18726 . 008Figure 3 . CORT is necessary and sufficient for increasing FSL . ( a ) CORT measurements in male mice subjected to stress are plotted against FSL . ( b ) Experimental timeline . ( c ) FSL following pre-treatment with the CORT synthesis inhibitor metyrapone in drinking water prior to and during isolation in females ( orange ) . The gray bar shows data from Figure 1 and is shown here for comparative purposes . ( d ) Experimental timeline . ( e ) Left bar shows FSL in male mice pretreated with Met prior to swim stress . Right bar shows FSL in male mice pretreated with vehicle prior to swim stress . ( f ) FSL is increased following incubation of slices from single males ( p<0 . 021 vs malesingle ) or group-housed females ( p=0 . 002 vs femalegroup ) with CORT . There is no significant difference in FSL between the two CORT-treated groups . DOI: http://dx . doi . org/10 . 7554/eLife . 18726 . 008 Next we investigated the underlying conductance that controls FSL in single-housed males and females . A rapidly activating , rapidly inactivating potassium ( K ) conductance contributes to FSL in a number of different cell types throughout the nervous system ( Meng et al . , 2011 ) . The channels responsible for this conductance are largely inactive at resting membrane potential , but this inactivation can be removed by a membrane hyperpolarization ( Yellen , 2002; Maffie and Rudy , 2008 ) . To test whether similar K channels contribute to the FSL in CRH neurons , we conducted experiments in which we did not deliver a membrane hyperpolarization prior to a depolarizing current step . The absence of this hyperpolarizing pre-pulse decreased the FSL in CRH neurons from socially-isolated females ( FSLnoHP: 37 . 8 ± 3 . 1 ms vs FSLHP: 55 . 3 ± 4 . 5 ms , paired t-test , p=0 . 0022 , Figure 4—figure supplement 1 ) . Rapidly activating and inactivating K currents are sensitive to millimolar concentrations of 4-aminopyridine ( 4-AP ) ( Alexander et al . , 2015 ) , and consistent with this , we observed shorter FSL in the presence of 2 mM 4-AP ( FSL4-AP: 36 . 8 ± 5 . 6 ms vs FSLcontrol: 62 . 8 ± 7 . 6 ms unpaired t-test , p=0 . 015 n = 8 , Figure 4a ) . We obtained voltage clamp recordings and characterized the currents responsible for regulating FSL ( malesingle n = 11 , femalesingle n = 12 ) . These currents were largely inactive at resting membrane potential , inactivated quickly and required membrane hyperpolarization to relieve inactivation ( Figure 4b ) . They were partially blocked by 2 mM 4-AP , fully blocked by 6 mM 4-AP ( n = 4 , Figure 4c ) , but were unaffected by a lower concentration of 4-AP ( 500 uM , data not shown ) ( Anderson et al . , 2010 ) . In addition , these currents were insensitive to 20 mM TEA ( data not shown ) . Neurons from socially-isolated females showed no difference in activation or recovery from inactivation in comparison to isolated males ( Figure 4d ) . There was no difference in current density in socially-isolated females when compared to socially-isolated males ( data not shown ) , arguing against an increase in channel number or conductance . We next asked whether channel kinetics might explain the differences in FSL . There was no difference in the activation kinetics between males and females , but currents from female mice had a longer decay time constant in comparison to males ( τfemale single: 55 . 8 ± 5 . 6 ms , n = 11 , vs τmale single: 41 . 9 ± 2 . 9 ms , n = 11 , unpaired t-test , p=0 . 019 , Figure 4e , f ) . These findings indicate that a slowing in the decay of a rapidly activating , rapidly inactivating voltage-gated K current is causative for the increase in FSL . 10 . 7554/eLife . 18726 . 009Figure 4 . Slower decay of 4-AP sensitive , voltage-gated K current in isolated females . ( a ) FSL is decreased by bath application of 2 mM 4-AP . ( b ) Voltage clamp traces from a male CRH neuron in response to different current step protocols as shown . ( c ) The rapidly activating and inactivating current was completely blocked by 6 mM 4AP . ( d ) The activation and inactivation curves are shown for single-housed females and males . ( e ) Currents at half-maximal activation , fit with a double-exponential are shown from males and females . ( f ) The slow inactivation time constant in malesingle and femalesingle is summarized ( unpaired t-test , p=0 . 02 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18726 . 00910 . 7554/eLife . 18726 . 010Figure 4—figure supplement 1 . FSL is sensitive to membrane hyperpolarization prior to depolarization . ( a ) Traces on left show voltage responses to a depolarizing current step with ( orange ) and without ( gray ) a hyperpolarizing pre-pulse . The absence of a hyperpolarizing pre-pulse decreases FSL as summarized in b ( n = 29 , p<0 . 01 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18726 . 010 Next , we asked whether these differences in the biophysical properties of the rapidly inactivating K current observed in socially-isolated females compared to males would be sufficient to alter the spike output of CRH neurons . We conducted experiments in which depolarizing current steps ( 20 pA intervals ) were preceded by a hyperpolarizing pre-pulse . CRH neurons from female isolated mice required a greater depolarization to generate a spike compared to CRH neurons from socially isolated males . This is revealed as a rightward shift in the relationship between spike probability and current step ( female: I50 = 49 . 5 ± 1 . 2 mV , n = 104 vs male: I50 = 55 . 0 ± 1 . 6 mV , n = 88 , p<0 . 0001 , K-S statistic ) . Finally , we compared the excitability of CRH neurons from isolated females and males . We used a protocol ( hyperpolarizing pre-pulse followed by a family of depolarizing steps ) that allowed for maximal activation of the rapidly activating K current described above . The rightward shift in the F-I plot indicates that CRH neurons from isolated females had lower excitability in comparison to isolated males ( p<0 . 0001 , K-S statistic , Figure 5b ) . 10 . 7554/eLife . 18726 . 011Figure 5 . Longer FSL decreases spike probability and neuronal firing . ( a ) Traces show firing properties of CRH neurons from individually housed male ( blue ) and female ( orange ) mice . Right panel shows the probability of eliciting a spike in response to varying current steps ( K-S statistic , p<0 . 0001 ) . ( b ) Responses from male and female CRH neurons to varying current steps . Right panel shows spike frequency in the first 50 ms of current step ( K-S statistic , p<0 . 0001 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18726 . 011
Our findings demonstrate that brief social isolation ( <24 hr ) affects the biophysical properties of PVN CRH neurons in a sexually dimorphic fashion . We report an increase in FSL and a decrease in neuronal excitability in socially-isolated female , but not in age-matched , male mice . By contrast , an acute physical stress ( swim ) increased FSL in group-housed females and single-housed males . In addition , social isolation occluded the effects of swim stress on female mice . CORT is both necessary for the isolation ( females ) and swim stress ( males and females ) induced increases in FSL and sufficient to increase FSL in the absence of stress . Finally , we noted the effect of social isolation was graded in female mice with paired mice exhibiting FSLs that were intermediate to those observed from group-housed or single mice . We examined the underlying conductance and report that it is sensitive to millimolar concentrations of 4-AP and insensitive to millimolar concentrations of TEA . In addition , FSL was profoundly sensitive to pre-hyperpolarizing pulses , which remove channel inactivation and make rapidly inactivating K channels available for opening in response to depolarizations . The features of these channels are consistent with Kv4 channels previously described in the PVN ( Lee et al . , 2012 ) . They do , however , exhibit a more hyperpolarized activation threshold in comparison to kinetically similar 4-AP sensitive channels reported in PVN parvocellular neurosecretory cells in rat ( Luther and Tasker , 2000 ) . We failed to note any differences in overall current density or voltage-dependent activation or recovery from inactivation . We did , however , observe an increase in the decay time constant of rapidly inactivating K currents in socially-isolated female mice compared to male mice , providing a potential underlying mechanism for the prolonged FSL in response to depolarization . The longer FSL we observed is not a classically defined characteristic of CRH neurons and , in fact , the absence of this delay has been used previously to categorize putative CRH neurons ( Hoffman et al . , 1991; Wamsteeker Cusulin et al . , 2013b ) . Our findings demonstrate that the biophysical characteristics of neurons are flexible and exquisitely sensitive to specific experiences . Whilst synaptic changes in the PVN ( Inoue et al . , 2013; Kuzmiski et al . , 2010; Wamsteeker Cusulin et al . , 2013a ) and other brain regions have been investigated , this is , to the best of our knowledge , one of the first reports of rapid intrinsic plasticity in neurons following acute stress . Our observations add to a number of observations that intrinsic ( synapse autonomous ) plasticity can have a profound effect on the output of neural networks ( Shah et al . , 2010; Kourrich et al . , 2015;O'Leary et al . , 2014 ) . In addition , as electrical fingerprints have been used to identify specific cell types , investigators should use caution when interpreting the data as the physiological or psychological state of the animal may have a dramatic impact on these fingerprints . Similarly , since neurons in the PVN can express multiple neuromodulators , caution must also be exercised when interpreting data from a genetically identified cell population . We have previously shown , using immunohistochemistry , that overlap between the genetically identified CRH cell population in PVN and non-CRH neurons is minimal ( <5% for vasopressin/CRH or oxytocin/CRH; <<1% for thyrotropin releasing hormone/CRH or somatostatin/CRH ) ( Wamsteeker Cusulin et al . , 2013b ) . These findings demonstrate that males and females react differently to some stressors but not others . Specifically , social isolation has definitive , CORT-dependent effects on CRH neurons in female , but not male mice . By contrast , both males and females show indistinguishable responses to an acute physical swim stress . These findings highlight the importance of carefully considering both the sex of the animal and the stressor modality when designing studies investigating neurobiological effects of stress . Indeed recent work demonstrating sex-specific expression of fear responses in rodents ( Gruene et al . , 2015 ) serves as a corollary to our observations and when taken together suggests that strategies for coping with stress are sex-specific . In particular , our findings highlight the importance of a social network in females and provides a neurobiological framework for further testing the underlying thesis of ‘tend and befriend’ strategy for females in response to stress ( Taylor et al . , 2000 ) . Finally , as our observations report sex-specific differences in response to stress in the first two weeks post-weaning , they provide clear evidence for biological effects on the stress axis that are independent of circulating gonadal hormones ( McCarthy and Arnold , 2011 ) . This exquisite sensitivity of the brain during the pre-adolescent period ( Foilb et al . , 2011 ) raises intriguing questions about the long-term consequences of subtle environmental and social manipulations on stress responses and behavior in males and females later in life .
All protocols received approval from the University of Calgary Animal Care and Use Committee in accordance with the guidelines of the Canadian Council on Animal Care guidelines ( Protocol # AC13-0027 ) . B6 ( Cg ) -Crhtm1 ( cre ) Zjh/J ( Crh-IRES-Cre ) mice and B6 . Cg-Gt ( ROSA ) 26Sortm14 ( CAG-TdTomato ) Hze/J ( Ai14 ) mice , whose generation has been detailed previously ( Madisen et al . , 2010; Taniguchi et al . , 2011 ) , were obtained from Jackson Laboratories ( stock number 012704 and 007914 respectively ) . These were maintained as colonies of homozygous mice , with one backcrossing to C57BL/6J background strain following their arrival . Genotyping was used to identify mutants using PCR procedures provided by the supplier . The following primers were used to identify Crh-IRES-Cre mutants: 5′-CTT ACA CAT TTC GTC CTA GCC and 5′- CAA TGT ATC TTA TCA TGT CTG GAT CC-3′ and ( 468 base pair resultant PCR band ) . To identify Ai14 mutants: 5′-GGC ATT AAA GCA GCG TAT CC-3′ and 5′-CTG TTC CTG TAC GGC ATG G -3′ were used ( 196 base pair band ) . The age of pre-adolescent mice ( post-natal day 21–35 ) was determined according to previous literature demonstrating that in C57/BL mice the onset of puberty occurs at approximately 5 weeks of age ( Nelson et al . , 1990; Mayer et al . , 2010 ) . Mice were individually housed on a 12 hr:12 hr light:dark cycle ( lights on at 7:00 ) with ad libitum access to food and water . Pairs of either homozygous Crh-IRES-Cre or Ai14 genotypes were mated , and the resulting heterozygous Crh-IRES-Cre;Ai14 offspring used in subsequent experiments . Sixteen hours prior to the acute stress protocol or slice preparation , mice were housed either individually , in same-sex pairs or same-sex groups ( 3–5 mice per group ) . Single or group -housed mice were randomly assigned to naive or stress conditions . For stress experiments , mice were exposed to a forced swim stress ( between 8:00 and 9:30 during the light phase ) consisting of 20 min in a glass cylinder ( 14 cm internal diameter ) filled with 30–32°C water . Following one hour of recovery in their home cage , mice were anesthetized and brain slices were prepared as described below . Experimental animals were anaesthetized with isoflurane and decapitated . The brain was quickly removed , and coronal brain slices ( 250 μm ) containing the PVN of the hypothalamus were obtained using a vibrating slicer ( Leica , Nussloch , Germany ) while submerged in ice cold slicing solution ( 0°C , 95% O2/5% CO2 saturated ) , containing ( in mM ) : 87 NaCl , 2 . 5 KCl , 0 . 5 CaCl2 , 7 MgCl2 , 25 NaHCO3 , 25 D‐glucose , 1 . 25 NaH2PO4 , 75 sucrose . Slices were then allowed a recovery period , of a minimum 60 min , in artificial cerebrospinal fluid ( aCSF ) ( 32 . 5°C , 95% O2/5% CO2 saturated ) containing ( in mM ) : 126 NaCl , 2 . 5 KCl , 26 NaHCO3 , 2 . 5 CaCl2 , 1 . 5 MgCl2 , 1 . 25 NaH2PO4 , 10 glucose . The CORT synthesis inhibitor , metyrapone was dissolved in polyethylene glycol and injected i . p . 60 min prior to swim stress at a dose of 75 mg/kg in a volume of 50 μL . For female isolation experiments , CORT was dissolved in the drinking water ( 500 μg/ml ) and given for 24 hr prior to isolation and during the entire isolation period . Hypothalamic slices were transferred to a recording chamber and superfused with 30–32°C aCSF at a flow rate of 1–2 ml/min . Slices were visualized using an upright microscope ( BX51WI , Olympus ) fitted with infrared differential interference contrast optics . CRH neurons were identified by their expression of tdTomato . Whole‐cell patch clamp recordings were obtained from CRH neurons using borosilicate glass microelectrodes with tip resistance between 2–5 MΩ . The normal intracellular solution contained ( in mM ) : 108 K‐gluconate , 2 MgCl2 , 8 Na‐gluconate , 8 KCl , 1 K2‐EGTA , 4 K2‐ATP , and 0 . 3 Na3‐GTP buffered with 10 mM HEPES . For images of filled cells , 0 . 2 mM Alexa-488 hydrazide and 10 mg·mL−1 biocytin were added to internal solution . Microscope images were captured using a Retiga EXi camera ( Qimaging ) and processed using ImageJ . Recordings were amplified using a Multiclamp 700B amplifier ( Molecular Devices , Union City , CA ) , low‐pass filtered at 1 kHz , and digitized at 10 kHz using the Digidata 1322 ( Molecular Devices ) . Data were recorded ( pClamp 9 . 2; Molecular Devices ) and stored on a computer for offline analysis . During all experiments , initial access resistance ( Ra ) was below 20 MΩ . Cell membrane properties were monitored for the duration of the experiments and only recordings in which changes to Ra Cm did not exceed 15% were accepted for analysis . For synaptic experiments , the cell membrane was voltage clamped at −80 mV . Spontaneous inhibitory ionotropic GABAA receptor postsynaptic currents ( sIPSCs ) were isolated by blocking AMPA‐ and kainate‐receptor‐mediated glutamatergic synaptic transmission with 6 , 7‐dinitroquinoxaline‐2 , 3‐dione ( DNQX , 10 μM ) . When measuring spontaneous excitatory postsynaptic currents ( EPSCs ) , the GABAA channel blocker picrotoxin ( 100 μM ) was included in the bath to isolate excitatory currents mediated by AMPA and kainate receptors . These spontaneous currents representing stochastic transmitter release were analyzed using MiniAnalysis 6 . 0 . 3 ( Synaptosoft , Decatur , GA ) . Event detection was set at three times the baseline noise and confirmed as synaptic events by eye . To determine RMP , cells were recorded in zero current ( I = 0 ) mode . Latency to spike initiation and firing thresholds were both measured using a current clamp depolarization step protocols . First , a baseline current injection that maintained the membrane voltage near −80 mV was chosen individually for each cell , which served to exclude the possible confounding influence of variable resting membrane potentials between cells . Next , a 200 ms/30 pA hyperpolarizing current injection was given followed by 250 ms/20 pA depolarizing current steps up to 140 pA . The latency time was measured as the duration from the point of initiating the depolarizing pulse to the initiation of the first spike . Firing threshold was determined as the membrane potential at the initiation of the first spike . Both firing threshold and membrane potential are corrected for a liquid junction potential of 12 mV , as calculated with solution ion concentrations . In experiments examining cell properties following 4-AP administration , baseline recordings were obtained , and then a ten-minute treatment period was allowed before secondary data were obtained . Synaptic currents were evoked by paired afferent stimulation ( every 5 s with an interstimulus interval of 50 ms ) and analyzed using Clampfit 9 . 2 ( Molecular Devices ) . Evoked postsynaptic current ( ePSC ) amplitudes were calculated from the baseline ( current before the first evoked response ) to peak of each evoked response . The paired pulse ratio ( PPR ) was calculated using the ratio of the amplitudes of the evoked pair ( peak 2/peak 1 ) from a minimum of a one-minute epoch within each cell . To isolate the voltage gated fast inactivating K current the following cocktail was applied to the slices for at least of 15 minutes prior recording: Bicuculline 10 µM , DNQX 10 µM , dAPV 50 µM , TTX 1 µM , TEA 20 mM . Each group represents a minimum of three animals for pharmacology experiments , or a minimum of four for stress experiments . Data points are presented as mean ± SEM . Statistical analyses for sIPSCs , and current clamp step data were performed in GraphPad Prism 4 using a one way ANOVA for multiple groups followed by a post-hoc Tukey’s multiple comparisons test; unpaired student's t-test were used for two group comparisons , and a K-S statistic for comparing two distributions . Drugs were dissolved into aCSF daily prior to experiments from frozen aliquots stored at −20°C and added to the bath by perfusion pump . The drugs were dissolved in accordance with guidelines either in DMSO , PEG , ethanol or distilled water . DNQX , 4-AP , d APV and metyrapone were obtained from Tocris ( Tocris Cookson , Ellisville , MO ) . Picrotoxin , bicuculline , TEA and corticosterone were obtained from Sigma ( Sigma‐ Aldrich , St . Louis , MO ) . TTX was obtained from Alomone labs ( Jerusalem BioPark ( JBP ) , Hadassah Ein Kerem , P . O . Box 4287 Jerusalem 9104201 , Israel ) .
Many species use social networks to buffer the effects of stress . The mere absence of a social network , however , may also be stressful . We examined neuroendocrine , PVN CRH neurons and report that social isolation alters the intrinsic properties of these cells in sexually dimorphic fashion . Specifically , isolating preadolescent female mice from littermates for <24 hr increased first spike latency ( FSL ) and decreased excitability of CRH neurons . These changes were not evident in age-matched males . By contrast , subjecting either males ( isolated or grouped ) or group housed females to acute physical stress ( swim ) , increased FSL . The increase in FSL following either social isolation or acute physical stress was blocked by the glucocorticoid synthesis inhibitor , metyrapone and mimicked by exogenous corticosterone . The increase in FSL results in a decrease in the excitability of CRH neurons . Our observations demonstrate that social isolation , but not acute physical stress has sex-specific effects on PVN CRH neurons .
Many species , including humans , use social interaction to reduce the effects of stress . In fact , the lack of a social network may itself be a source of stress . Recent research suggests that young girls are more sensitive to social stress than boys . This could mean that social networks are more important for females in general , and that young females from different species , such as mice , may be more sensitive to social isolation than males . However , few studies have examined how social isolation affects the brain cells that control the release of stress hormones . As such , it remains unknown whether isolating individuals from their social group impacts on the brain in sex-specific ways . Senst , Baimoukhametova et al . now show that the brains of young male and female mice react differently to social isolation . Less than a day after separation from their littermates , the activity in the brain cells of female mice became markedly different from that of isolated males . In contrast to social isolation , the physical stress of being made to swim produced similar changes in the brains of both male and female mice . Further experiments then showed that the changes in the brain cells that control the release of stress hormones required a signalling chemical called corticosterone , which is produced in response to stressful situations . This suggests that , in repsonse to soical isolation , the females are experiencing more stress than the males . Following on from this work , one future challenge will be to investigate if reuniting a social group erases the effects of social isolation on the brain . Further experiments could also examine the behavioural and physiological effects of social isolation , including how females respond to later stressful events .