contains high-power photomicrographs of a 5-HT- and a non-5-HT-containing cell in the DRN. The left panel contains biocytin-labeled cells, the middle panel is the TPH-stained cells, and the right panel contains the merged images.
Figure 1 Immunohistochemical identification of 5-HT- and non-5-HT-containing neurons. Immunofluorescent photomicrographs of recorded neurons filled with biocytin (red) and co-stained for tryptophan hydroxylase (TPH) (green). The merged image is depicted in the (more ...)
LCM and RNA Analysis
LCM techniques were implemented to isolate TPH-positive cells from the heterogeneous cell population in the DRN. Following microdissection, RNA was extracted from 5-HT-containing cells in the DRN of SD and WKY rats. represents a subpopulation of measurements taken from the two strains. Values are listed as the fraction of RNA against β-actin, percentage change from SD to WKY as well as p-values for unpaired t-tests. We focused our analysis on mRNA that encode for proteins known to have a role in stress responses, anxiety, depression, or addiction, and also those critical for regulating the excitability of DRN neurons. In 5-HT neurons in the DRN of WKY rats, there was a significant reduction (66% decrease) in mRNA encoding the predominant isoform of TPH expressed in the brain, TPH2, compared with SD rats. Differences in 5-HT receptor mRNA included that encoding for 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT6, and 5-HT7A. Notably, 5-HT neurons in the DRN of WKY rats demonstrated a significant reduction in 5-HT1A and 5-HT6 receptor mRNA (21 and 8%, respectively), and there was an 87% increase in 5-HT7A mRNA. The CRF R1 receptor mRNA expression was not significantly different based on strain; however, there was a 54% increase in CRF R2 mRNA expression in WKY rats. There was relatively little expression of mineralocorticoid receptor (MR) mRNA in the DRN. Moreover, although glucocorticoid receptors (GRs) were expressed in the DRN, there was not a significant difference in expression between strains. Finally, there was a significant reduction (12%) of GABAA α1 subunits and remarkably an 88% reduction in GABAA β3 subunits in 5-HT neurons of WKY rats.
Normalized aRNA Levels That Encode for Anxiety- and Depression-Related Proteins in 5-HT DRN Neurons of SD vs WKY Rats
Electrophysiological studies were focused on receptor-mediated responses with known functional effects that have been well characterized in either the DRN or other regions, namely, 5-HT1A, CRF R1 and R2, and GABAA subunit effects. Electrophysiological data were recorded from both 5-HT- and non-5-HT-containing neurons and results are reported for both cell types in the text and tables; however, only data from 5-HT neurons are shown graphically.
Cellular Characteristics of 5-HT and Non-5-HT Cells in the DRN of SD and WKY Rats
Using current clamp configuration, cellular characteristics were recorded from 5-HT- and non-5-HT-containing cells in the DRN of SD and WKY rats. contains the summary data (means±SEM). The cellular characteristics of the 5-HT and non-5-HT neurons in SD rats were similar to those previously reported (Beck et al, 2004
). However, 5-HT cells of WKY rats had a more hyperpolarized resting membrane potential (RMP) when compared with SD 5-HT cells (p
<0.05), suggesting a decreased intrinsic excitability of WKY 5-HT neurons. Interestingly, there were no differences in either membrane input resistance or tau between SD and WKY 5-HT neurons. The response to 5-HT1A receptor activation was also assessed. A saturating concentration of the 5-HT1,7 receptor agonist 5-CT (100
nM) was bath applied to assess the hyperpolarization (mV) associated with 5-HT1A receptor activation (Beck et al, 2004
). Based on the mRNA data, we hypothesized that 5-HT neurons of WKY rats would show an attenuated response to 5-HT1A receptor activation. The response to 5-CT administration was not different between SD and WKY rats for both 5-HT- and non-5-HT-containing neurons in the DRN. However, using a criterion of 5
mV hyperpolarization minimum, there was a trend for a difference in the percentage of neurons exhibiting a 5-HT1A receptor-mediated response, that is, 85% for neurons recorded from SD rats, but only 75% for neurons from WKY rats. It is possible that a nonsaturating concentration of 5-CT would reveal a more substantial difference between strains. Additionally, there was a difference between the non-5-HT neurons, with 50% of the neurons from SD rats exhibiting a response, but only 20% of the neurons from the WKY rats. The χ2
analysis revealed that the data were not significant (p
Cellular Characteristics and 5-CT Response of 5-HT and Non-5-HT Neurons in the DRN of SD and WKY Rats
Functional Effects of GABAA Subunit Changes in WKY 5-HT Neurons
The rise and decay kinetics of the mIPSC are entirely dependent on the GABAA
receptor subunit composition. The RNA data indicated only a 12% reduction in α
1 subunit expression, yet demonstrated an 88% reduction in β
3 subunit expression in WKY rats. Based on previous studies from β
mice (Ramadan et al, 2003
), we hypothesized that mIPSCs recorded from 5-HT neurons in the DRN of WKY rats would have significantly faster decay kinetics than SD rats. The baseline data from all of the experiments were combined for the analysis of baseline mIPSC parameters. There were no significant differences in baseline mIPSC characteristics between experiments from the same cell type and strain. The mIPSC parameters from SD and WKY 5-HT and non-5-HT neurons are presented in . A one-way ANOVA with post hoc t
-test revealed that non-5-HT neurons in SD rats exhibited significantly larger mIPSC amplitude compared with all other groups. However, this was the only significant difference in basal mIPSC properties. Surprisingly, there were no significant differences specifically in rise or decay kinetics in the mIPSC measured between SD and WKY 5-HT neurons. There were also no significant differences in mIPSC frequency or amplitude between 5-HT neurons based on rat strain, suggesting a similar probability of release of GABA from presynaptic terminals as well as similar basal receptor occupancy/receptor density.
mIPSC Characteristics for 5-HT and Non-5-HT Neurons in the DRN of SD and WKY Rats
The β3 subunit is important in the activation of the receptor, and therefore the difference between SD and WKY rats may manifest when the receptor is activated by selective agonists. To confirm the mRNA data, we used standard immunohistochemistry protocols to assess β3 protein expression on 5-HT neurons. Consistent with the quantitative mRNA data, qualitative inspection of changes in GABAA β3 immunohistochemistry in SD and WKY rats revealed an overall reduction in fluorescent intensity and β3-positive cells in WKY animals (Supplementary Figure S1). Despite the fact that we did not see a change in basal mIPSC kinetics between SD and WKY 5-HT neurons, it is possible that this difference in GABAA receptor subunit expression would manifest as a change in pharmacological efficacy.
To test the hypothesis that there was a functional effect of GABAA β
3 reduction in WKY rats, loreclezole, an anticonvulsant and GABAA
modulator that selectively binds to β
2/3 subunits, was bath applied. We reasoned that as there was no change in β
2 mRNA expression, the potential differences in loreclezole (10
μM)-mediated mIPSC prolongation would be because of alterations in β
3 expression. Loreclezole experiments were conducted only in 5-HT neurons. depicts representative traces before and after application of loreclezole in 5-HT neurons of SD and WKY rats. Loreclezole did not significantly increase mIPSC frequency in SD or WKY rats, nor did it significantly augment mIPSC amplitude (N
=7 for both SD and WKY; Supplementary Table S1 and ). Loreclezole significantly prolonged mIPSC decay only in SD 5-HT neurons, but not in WKY 5-HT neurons (Supplementary Table S1 and ). Based on these data, we concluded that the large reduction in GABAA β
3 mRNA in WKY 5-HT neurons results in a functional decrease in GABAA
Figure 2 Response to loreclezole is absent in 5-HT neurons of WKY rats. (a) A representative averaged mIPSC and trace (inset) from a 5-HT neuron in a SD (left) or WKY (right) rat before and after 10μM loreclezole application, an anti-convulsant (more ...)
The alteration in β3 function may have implications for the functional effects of the α1 subunit, although only a small change in α1 subunit expression itself was found. In hippocampal cultures from β3–/– mice, there was a significant enhancement in the efficacy of zolpidem that allosterically binds selectively to α1 containing GABAA channels, because of loss of α2/3 subunit expression at the cell surface. Similar to β3–/– mice, we found a zolpidem-elicited increase in mIPSC amplitude in 5-HT neurons of WKY rats compared with SD rats (Supplementary Table S1).
CRF Receptor Activation Effects on mIPSCs in Cells Recorded from the DRN of WKY Rats
In a recent paper from our laboratory we demonstrated that CRF R1 activation enhances GABAergic synaptic transmission both pre- and post-synaptically in 5-HT-containing neurons in the DRN of SD rats (Kirby et al, 2008
). Additionally, in both 5-HT and non-5-HT neurons, CRF produced a small, yet physiologically relevant, inward current, through activation of different receptors: CRF R2 in 5-HT neurons and CRF R1 in non-5-HT neurons. It has been demonstrated that WKY rats are behaviorally similar to rats that have been previously exposed to stressors and, therefore, we predicted that there would be an attenuation of the in vitro
CRF effects on GABAergic synaptic transmission and cell excitability established by Kirby et al (2008)
. However, in light of the mRNA data, we further predicted that some, if not all, of the CRF R2 effects may be retained as there was a significant upregulation of CRF R2 mRNA in 5-HT neurons of WKY rats.
The same protocol and concentrations of oCRF (selective R1 agonist) and Ucn II (selective R2 agonist) were used as described in Kirby et al (2008)
. Data were collected from WKY rats in parallel using the same drugs and stock solutions. Therefore, the data from the SD rats are the controls for the data obtained from the WKY rats (see Materials and Methods).
displays representative traces (left) and the averaged mIPSC (right) recorded from cells before and after the administration of oCRF from SD 5-HT (upper panel) and WKY 5-HT (lower panel) neurons. contains summary graphs of the mIPSC frequency () and amplitude () following oCRF bath application for 5-HT neurons recorded in the DRN of SD and WKY rats (N=26 and 19, respectively). In direct contrast to our findings in SD rats, oCRF did not increase mIPSC frequency or amplitude in neurons recorded from WKY rats (Supplementary Table S2 and ). Interestingly, in non-5-HT WKY neurons, oCRF did significantly increase both mIPSC frequency and amplitude (p<0.05, N=7); these effects were not apparent in non-5-HT neurons in SD rats (Supplementary Table S2).
Figure 3 CRF responses are attenuated in 5-HT DRN neurons of WKY rats. (a) Representative traces (left) and averaged mIPSC (right) from recordings made from 5-HT neurons of SD (top) and WKY (bottom) rats before and after 10nM oCRF bath application. (b) (more ...)
In a separate set of experiments, the selective CRF R2 agonist, Ucn II, was bath applied to the slice. contains representative traces (left) and averaged mIPSCs before and after Ucn II administration in recordings made from 5-HT neurons in SD (upper panel) and WKY (lower panel) rats. In SD rats, Ucn II had no effect on frequency but significantly increased the amplitude of mIPSCs (p
=17; and Supplementary Table S2). Similarly to what was reported in SD rats, Ucn II had no effect on mIPSC frequency in WKY 5-HT neurons (N
=13; ). Interestingly, similarly to SD 5-HT neurons, Ucn II significantly augmented mIPSC amplitude in WKY 5-HT neurons (). Kirby et al (2008
) reported no effect of Ucn II on mIPSC frequency or amplitude in non-5-HT-containing neurons of SD rats. However, Ucn II did significantly augment mIPSC amplitude in WKY non-5-HT neurons (p
=8; Supplementary Table S2).
Figure 4 The effects of the selective CRF R2 agonist Ucn II on mIPSC amplitude were retained in WKY rats. (a) Representative traces (left) and averaged mIPSC (right) from recordings made from 5-HT neurons of SD (top) and WKY (bottom) rats before and following (more ...)
Therefore, in the WKY stress-hyperresponsive rats, the CRF R1-mediated increase in frequency was attenuated in 5-HT neurons, but enhanced in non-5-HT neurons. Ucn II significantly increased mIPSC amplitude in WKY 5-HT-containing cells. In WKY non-5-HT neurons, both oCRF and Ucn II augmented mIPSC amplitude. Thus, in the DRN 5-HT neurons of stress-hyperresponsive rats, the CRF R2-mediated response on GABAergic synaptic transmission was retained, but the CRF R1 response eliminated. Moreover, CRF modulation of GABAergic transmission that was not seen in SD non-5-HT neurons was significantly enhanced in non-5-HT neurons in the WKY rats.
This difference in CRF response between strains is specific to the DRN. In a set of control experiments conducted in the neighboring ventral serotonergic structure, the MRN, CRF was found to augment mIPSC amplitude in 5-HT cells of both SD and WKY rats (Supplementary Figure S2). Despite evidence that this structure is also involved in affective behavior, CRF responsiveness of these cells was retained in WKY rats, suggesting a regional specificity as opposed to a global change.
oCRF and Ucn II Do Not Alter the Inward Current in WKY Rats
In SD animals, oCRF and Ucn II significantly increased the inward current in 5-HT-containing cells. Pretreatment with the CRF R2 antagonist ASVG-30, but not the CRF R1 antagonist antalarmin, is able to block the effect of oCRF on inward current. This evidence suggests that this effect is mediated by the CRF R2 receptor (Kirby et al, 2008
). Only oCRF, but not Ucn II, significantly increased the inward current in non-5-HT cells in an antalarmin-sensitive manner, suggesting that in this cell type CRF R1 receptors mediate the effect. In this study, the effects of oCRF or Ucn II on inward current were not apparent in either 5-HT- or non-5-HT-containing cells of WKY rats. The CRF R1- and CRF R2-mediated increase in inward current was absent in non-5-HT- and 5-HT-containing neurons from WKY rats, respectively. Thus, the net effect of CRF receptor activation on 5-HT neurons in WKY rats was selective to an enhancement of GABAA
receptor-mediated currents, without a simultaneous increase in GABA release.
The GABAmimetic Effects of Ethanol (EtOH) on 5-HT Neurons in DRN of SD and WKY Rats
Along with demonstrating a global stress hyperresponsivity, WKY rats also show an increase in alcohol preference (Pare et al, 1999
). EtOH depresses DRN firing rates in vivo
and presumably depresses 5-HT release in the forebrain (Pistis et al, 1997
). It is possible that DRN dysregulation and subsequent abnormal physiological responses to EtOH in the DRN may be in part responsible for the behavioral increase in alcohol preference documented in WKY rats. Physiologically relevant concentrations of EtOH, 10–100
mM, have GABAmimetic effects via enhancement of mIPSC frequency or amplitude in in vitro
slices made of limbic regions such as the central amygdala (Nie et al, 2004
). Moreover, EtOH-mediated effects can be blocked by pretreatment of the CRF R1 selective antagonist antalarmin and is also not apparent in CRF R1−/−
mice. We reasoned that it was possible, given the alterations in GABAA
subunit expression and pharmacological sensitivity as well as attenuation of CRF R1-mediated enhancement of GABAergic synaptic transmission, that a midrange concentration of EtOH (50
mM) would have disparate effects on GABAA
mIPSC frequency and amplitude in 5-HT neurons from SD vs
WKY rats. We also wanted to determine if antalarmin pretreatment would have a similar block on the GABAmimetic effects of EtOH. shows representative traces from 5-HT neurons recorded from SD () and WKY () rats. In a separate set of SD rats, the slice was pretreated with antalarmin (300
nM) following a baseline mIPSC recording, but before EtOH treatment. Similar to what was reported in our previous work, there was a trend for antalarmin to increase mIPSC amplitude (p
=0.08). contains summary bar graphs of the change in frequency and amplitude from baseline following EtOH (50
mM) bath application. We found that EtOH (50
mM) significantly enhanced mIPSC frequency and amplitude (p
<0.05 in both SD and WKY DRN 5-HT neurons, N
=10 and 9, respectively), yet there was no significant effect of strain (two-way repeated-measures ANOVA, p
>0.05; Supplementary Table S2 and ). Pretreatment with Antalarmin had no effect on EtOH-mediated enhancement of mIPSC frequency, but did attenuate the EtOH effect on mIPSC amplitude (one-way repeated-measures ANOVA, p
<0.05, baseline vs
>0.05, antalarmin vs
=6). This finding is consistent with other studies demonstrating an antalarmin block of EtOH GABAmimetic effects. It is also suggested that there were two different mechanisms underlying the EtOH effects on presynaptic release of GABA vs
postsynaptic enhancement of the amplitude. However, based on our comparison of SD with WKY rats in this set of experiments, we conclude that there is no appreciable difference in the EtOH response in this nucleus in stress-hyperresponsive rats.
Figure 5 GABAmimetic effects of EtOH (50mM) were not different in 5-HT neurons from SD and WKY rats. (a) Representative traces from recordings made from 5-HT neurons of SD (ai, aii) and WKY (aiii) rats before and after application of 50mM EtOH. (more ...)