Co-localization of EGFP with TH in TH-EGFP mice
We used transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the tyrosine hydroxylase (TH) promoter to identify NTS CA neurons. In agreement with what we had previously reported (Appleyard et al., 2007
) we found that overall TH co-localized with EGFP in 84% of NTS neurons from the TH-EGFP mice. However, we did find that the co-localization varied from region to region, for example more caudal NTS EGFP neurons were extensively co-localized with TH (90–100%), while the more rostral NTS neurons were less co-localized (50–70%). Co-localization was high (>84%) in the regions in which our recordings are made, within 200 μm rostral or caudal from obex and medial to the ST. We did observe regions of very low co-localization, particularly in the dorsal motor nucleus of the vagus (DMNV, 10N), which contained cells that expressed EGFP, but only a low percentage (24%) of which were immunopositive for TH. However, we do not record from these neurons in our studies. All TH-EGFP neurons could be easily visualized and identified for recordings. As an additional confirmation that the neurons we were recording from expressed TH we also performed post-hoc staining by including neurobiotin in our pipette. We found that neurobiotin/strepavidin was co-localized with both EGFP and TH immunostaining in 5/6 (83.3%) neurons examined.
Ghrelin inhibits spontaneous glutamate inputs onto TH-EGFP neurons
Application of 100nM ghrelin increased the inter-event interval of spontaneous glutamate inputs (sEPSCs) in 16out of 26TH-EGFP neurons tested (P<0.05; KS test, ). The basal rate of sEPSCs is highly variable between TH-EGFP neurons with the frequency ranging from 0.5 to 12.6 Hz, as has been reported previously (Appleyard et al., 2007
). However, across responsive neurons, ghrelin decreased the sEPSC frequency from 5.2±1.1 Hzin control (ACSF) to 3.1 ± 0.7 Hz (ghrelin) (n=16), with an average inhibition of 37 ± 3% that was partially reversed following a 10 minute wash to 24 ± 5% (, n=15). In contrast, ghrelin did not consistently change the amplitude of the sEPSCs (; Control: −54.3 ± 3.4 pA vs. Ghrelin: −52.0 ± 3.6 pA). The average frequency of the non-responsive neurons was 1.9 ± 0.4 Hz (Control) and 2.0 ± 0.5 Hz in ghrelin (n=10), significantly lower than the ghrelin responsive neurons (p<0.05, Student’s test).
Figure 1 Ghrelin inhibits spontaneous EPSCs (sEPSCs) frequency in TH-EGFP neurons; an effect blocked by the GHSR antagonist D-Lys-3-GHRP-6. A. Visualization of the NTS horizontal brain-slice preparation from a TH-EGFP mouse using DIC (left) and florescence (right). (more ...)
The GHSR receptor mediates ghrelin’s inhibition of the frequency of sEPSCs
Pre-incubation with the GHSR1 antagonist D-Lys-3-GHRP-6 (30uM) completely attenuated the effects of 100 nM ghrelin in 8 out of 9 TH-EGFP neurons (; P>0.05, KS test). Across neurons, this constituted a significant block of ghrelin’s effects (P<0.05, Fisher exact test, n=1/9 and n=16/26), with the average sEPSC frequency being 4.7 ± 1.0 Hz in control, 3.8 ± 0.8 Hz in D-Lys-3-GHRP-6 and 3.9 ± 1.0 Hz in D-Lys-3-GHRP-6 + ghrelin. There was no significant effect of D-Lys-3-GHRP-6 or D-Lys-3-GHRP-6 + ghrelin on the sEPSC amplitude across neurons (, n=8, p>0.05, Student’s t-test; Control = −41.6 ± 4.4 pA, D-Lys-3-GHRP-6 = −39.3 ± 4.5 pA, D-Lys-3-GHRP-6 + ghrelin = −38.3 ± 3.9 pA). D-Lys-3-GHRP-6 alone had no significant effect on the inter-event interval in 6/8 TH-EGFP neurons (KS test p>0.05), but significantly increased the inter-event interval in 2 neurons (KS test p<0.05). Although the underlying mechanism is not clear, this drug has been reported to have other actions in addition to its antagonism of the GHSR1 (Schioth et al., 1997
; Depoortere et al., 2006
; Erriquez et al., 2009
Ghrelin inhibits ST-EPSCs in TH-EGFP neurons in a concentration-dependent manner
While there are several sources of glutamate inputs in the NTS, a major source is the solitary tract (ST) afferent fibers, including those coming from the GI tract. Cutting the brainstem slices horizontally preserves a lengthy segment of the ST in the same plane as the cell bodies of NTS (). This allows placement of the stimulating electrode on the visible ST at a sufficient distance from the recording area to minimize focal activation of local interneurons and interconnecting fibers. Brief shocks (100 μsec duration) passed through the stimulating electrode evoked excitatory postsynaptic currents (ST-EPSCs). As we have described previously ST-EPSCs in TH-EGFP neurons had nearly invariant latencies, few failures, frequency-dependent amplitude depression and were attenuated by the non-NMDA glutamate antagonists NBQX or CNQX (Appleyard et al., 2007
). Bath application of 100 nM ghrelin significantly inhibited the amplitude of the ST-EPSC in 10/11 neurons tested (), from −237 ± 24pA in ACSF to −170 ± 21 pA in ghrelin, an effect partially reversed by wash to −197.4 ± 22pA (). The range of inhibition was from 10% to 63%, with the average being 30 ± 5% (, n=10). In contrast, continued perfusion in ACSF did not significantly change the ST-EPSC amplitude; control = −240 ± 37 pA vs 10 minute continued perfusion in ACSF = −241 ± 37 pA (, 0nM dose, n=9). Ghrelin did not significantly change either the input resistance (Responders: Control = 569±91MΩ,100 nM ghrelin = 610 ± 105 MΩ, n=10) or holding current (Responders: Control = −11.9 ± 1.1 pA, 100 nM ghrelin = −11.0 ± 1.1 pA, n=10) in TH-EGFP neurons consistent with ghrelin’s actions being predominately pre-synaptic.
Figure 2 Ghrelin inhibits the amplitude of ST stimulated EPSCs in TH-EGFP neurons. A. Representative trace of two ST-stimulated EPSCs. ST activation evoked monosynaptic EPSCs in TH-EGFP neurons. VM = −60 mV. Ghrelin significantly inhibited the amplitude (more ...)
The effects of ghrelin were concentration dependent with 10 nM ghrelin producing a 20.4 ± 2% inhibition in 5/7 TH-EGFP neurons (ST-EPSC amplitude control = −244 ± 32 pA, 10nM ghrelin = −196 ± 27 pA, n=5) and 30 nM ghrelin producing a 25.5 ± 2% inhibition in 6/9 neurons (ST-EPSC amplitude control = −174 ± 16 pA, 30nM ghrelin = −130 ± 13 pA, n=6); compared to 100 nM ghrelin producing the 30 ± 5% inhibition in 10/11 neurons described above (; ACSF vs. 100 nM ghrelin p<0.001; ACSF vs. 30nM ghrelin p<0.001; ACSF vs. 10 nM ghrelin p<0.005, One way ANOVA). The EC50 for ghrelin’s inhibition of ST-EPSCs in TH-EGFP neurons was approximately 7nM.
Presynaptic actions of ghrelin on NTS-CA neurons
Ghrelin significantly increased the paired-pulse ratio (EPSC2 amplitude/EPSC1 amplitude when two shocks were applied 20 mS apart) in ghrelin responsive neurons. On average ghrelin increased the size of the PPR across neurons from 0.65 ± 0.05 in control (ACSF) to 0.82 ± 0.09 in ghrelin (p<0.05, Student’s t-test, n=10). A change in the paired pulse ratio is normally associated with a pre-synaptic mechanism of action, especially as post-synaptic AMPA receptor desensitization has little effect on the peak amplitude of ST-EPSCs in NTS neurons (Chen et al., 1999
). To study this more closely we examined mini-EPSCS (mEPSCs) in the presence of 2uM TTX to block action potentials (). The voltage was held at −60mV, the approximate reversal potential for chloride to isolate glutamatergic mEPSCs. Ghrelin significantly increased the inter-event interval in 7/8 NTS TH-EGFP neurons (, p<0.05 KS test), with an average frequency of 2.0 ± 0.3 Hz in TTX only to 1.4 ± 0.1 Hz in TTX + ghrelin, (n=7) and an average inhibition of 40 ± 5% (). In contrast, ghrelin had no consistent effect on mEPSC amplitude (; TTX only = −37.3 ± 3.6 pA; TTX + ghrelin = −36.9 ± 3.4 pA). Taken together with the lack of effect of ghrelin on either input resistance or holding current these data are consistent with ghrelin’s actions being predominately pre-synaptic.
Figure 3 Ghrelin significantly decreased the frequency of miniature EPSCs in TH-EGFP neurons. A. Representative traces of mEPSCs from control (ACSF) conditions and in the presence of 100nM ghrelin. B. A graph showing the frequency of mEPSCs over time. Ghrelin (more ...)
Figure 4 Ghrelin decreases the firing rate of TH-EGFP neurons as well as ST-stimulated APs. A. Representative trace from a current clamp experiment showing the firing rate of a TH-EGFP neuron. Bath application of ghrelin (100nM) significantly reduced the basal (more ...)
Ghrelin inhibits the basal firing rate of TH-EGFP neurons
As reported previously we found that the majority of TH-EGFP neurons fire at rest (Appleyard et al., 2007
). Bath application of 100nM ghrelin significantly reduced the basal firing rate in 5/6TH-EGFP neurons from 2.6 ± 1.0 Hz to 0.9 ± 0.7Hz, with the average inhibition being 82.5 ± 10% (, p<0.01, Student’s t-test, n=5). This effect was partially reversed following wash to 56.3 ± 14%. To determine whether ghrelin inhibited the firing rate of TH-EGFP neurons through its effect to reduce excitatory glutamate inputs onto these neurons we examined whether the effects of ghrelin were attenuated by the glutamate antagonist NBQX. 20 μM NBQX alone inhibited the firing rate of 6/7TH-EGFP neurons from 4.2 ± 1.0 Hz to 3.2 ± 0.9 Hz (p<0.01, Student’s t-test, n=7), suggesting that spontaneous excitatory glutamate inputs do indeed alter the firing rate of these neurons, at least in our slice preparation. NBQX completely blocked the effects of ghrelin in 6/7neurons (, p>0.05, Student’s t-test). Across neurons NBQX blocked the effect of ghrelin on the firing rate in TH-EGFP neurons (p<0.05, Fishers exact test), with an average firing rate across neurons of 3.2 ± 0.9 Hz in NBQX alone and 3.1 ± 0.9 Hz when NBQX and ghrelin were co-applied. This data supports the hypothesis that ghrelin’s effects are primarily due to an inhibition of glutamate inputs onto these neurons. We also found that when ghrelin was applied alone it hyperpolarized 3/6 neurons an average of 6.8 ± 1.3 mV (including the example shown in ), while no effect was seen in the other 3 neurons (Average change 0.4 ± 1.9 mV). However, ghrelin did not significantly change the membrane potential of any NTS TH-EGFP neurons tested when applied in the presence of NBQX (Control = −59.7 ± 1.3 mV, NBQX = −59.9 ± 1.0 mV, Ghrelin + NBQX = −58.5 ± 1.5 mV; n=7, p>0.05, Student’s t-test). As ghrelin did significantly reduce the firing rate of one TH-EGFP neuron in the presence of NBQX from 5.0 Hz to 2.8 Hz we cannot rule out an additional effect of ghrelin in a small population of TH-EGFP neurons.
Ghrelin inhibits ST-evoked APs in TH-EGFP neurons
To test whether ghrelin also inhibits the ability of ST afferents to generate an action potential in TH-EGFP neurons we measured the success rate or the input output ratio (I-O) of ST stimulation to action potential firing in TH-EGFP neurons. Under control conditions (ACSF) the I-O ratio was 0.87 ± 0.13 (n=11). Ghrelin significantly reduced the I-O ratio to 0.38 ± 0.14 in 7/11 TH-EGFP neurons (, p<0.01, Student’s t-test). This effect was partially reversed following wash (I-O ratio = 0.66 ± 0.12; p>0.05 from control, Student’s t-test, n=7).
Ghrelin inhibits ST-EPSCs in the minority of non-florescent NTS neurons from the TH-EGFP mice or POMC-EGFP NTS neurons
100nM ghrelin inhibited ST-EPSC amplitude in 3/9 non-green florescent neurons in the NTS of TH-EGFP mice. The average ST-EPSC amplitude in the responders was −382 ± 113 pA in control and −305 ± 94 pA in ghrelin, with an average inhibition of 20 ± 3% (n=3). The average ST-EPSC amplitude in non-responders was −246 ± 26 pA in control compared to −251 ± 28 pA in ghrelin (101±1% of control, n=6). Ghrelin had no significant effect on input resistance in EGFP negative neurons, even in the 3 responsive neurons (Control: 520 ± 19 mΩ, ghrelin: 496 ± 5 mΩ, n=3).
We next examined the effect of ghrelin on another population of NTS neurons the pro-opiomelanocortin (POMC) neurons, which were indentified using a POMC-EGFP transgenic mouse (Cowley et al., 2001
). We have previously demonstrated that 80% of POMC-EGFP neurons receive direct strong inputs from ST-afferents and that CCK increases glutamate inputs onto these neurons (Appleyard et al., 2005
). However, we found that 100nM ghrelin only had a significant effect in 2/9 POMC-EGFP neurons with the average ST-EPSC amplitude being −130 pA in control and −106 pA in ghrelin, an average inhibition of 19% (n=2). In non-responders the average amplitude was −236 ± 49 pA in control and −225 ± 49 pA in ghrelin (n=7). Again, ghrelin had no significant effect on input resistance (Control = 589 ± 60 mΩ, ghrelin = 571 ± 56 mΩ, n=9). These results demonstrate that ghrelin preferentially inhibits ST-EPSCs in TH-EGFP neurons over either non-TH or POMC-EGFP NTS neurons (TH vs. non TH p<0.05; TH vs. POMC p<0.01; Fisher’s exact test).
Ghrelin affects both CCK-sensitive and insensitive NTS TH-EGFP neurons
CCK has been shown to increase c-fos activation of NTS TH immunopositive neurons (Monnikes et al., 1997
; Willing and Berthoud, 1997
; Rinaman et al., 1998
) and we and others have shown that CCK increases glutamate inputs onto TH neurons (Baptista et al., 2005
; Appleyard et al., 2007
). As ghrelin significantly decreased the frequency of sEPSCs, the opposite effect to CCK, we determined whether ghrelin inhibited spontaneous glutamate inputs onto TH-EGFP neurons that were also sensitive to CCK. Ghrelin significantly inhibited sEPSCs onto 4/6 CCK-sensitive TH-EGFP neurons an average of 32 ± 9% (: CCK control: 1.3 ± 0.6 Hz, CCK: 6.6 ± 2.2 Hz; ghrelin control: 2.0 ± 0.9 Hz, ghrelin: 1.1 ± 0.5 Hz, p<0.05 for both CCK and ghrelin, KS test, n=4). Ghrelin also inhibited the sEPSC frequency in 3/5 CCK-insensitive TH-EGFP neurons by an average of 44 ± 1% (CCK Control 2.1 ± 0.8 Hz, CCK 2.1 ± 0.9 Hz; ghrelin control 3.8 ± 2.6 Hz, ghrelin 2.2 ± 1.5 hz, p<0.05 for ghrelin but not CCK, KS test, n=3).
Figure 5 Ghrelin inhibits sEPSC frequency in CCK-sensitive and insensitive TH-EGFP neurons. A. Representative traces from control conditions (ACSF), following a bath application of ghrelin (100nM) and a subsequent application of CCK (100nM) in the same neuron. (more ...)
Fasting increases the size of ghrelin’s effects in NTS TH-EGFP neurons
Animals and people tend to feel hungrier and eat more in a fasted state. To examine whether the effects of ghrelin on the incoming afferents are increased following a fast, mice were either food-deprived for 18 hours (fasted) or maintained with ad lib access to food (fed), with both groups having ad lib access to water. Glucose concentrations are quite high in our normal ACSF at 10–15mM (10 mM glucose with additional glucose added to adjust the osmolarity of our solutions), which is fairly standard for brain slice recordings. However, given that both plasma and CSF glucose levels are generally lower and are further reduced during fasting (For review see Routh, 2002
) we lowered the concentration of glucose we use in our ACSF solution to 5 mM for these experiments for slices from both fasted and fed animals in order to be closer to the physiological range(with sucrose substituted to maintain osmolarity). 5 mM glucose is still on the high side of physiological levels for fasting, even for plasma concentrations (Routh, 2002
); however, it was the lowest concentration of glucose that maintained the health of the slices for the duration of the experiments. 5 of these experiments in each condition (10 total) were performed with a blind design, where the experimenter did not know whether the slices were from a fed or fasted animal. We found no significant difference between the results when the investigator was blind to the treatment group compared to when they knew the treatment group and so the results were combined. Ghrelin significantly inhibited the ST-EPSC amplitude in 8/12 neurons from −222 ± 33 pA to −171 ± 33 pA in slices taken from fed animals and maintained in ACSF with 5 mM glucose, an average inhibition of 26 ± 5%. In contrast, in neurons recorded from slices taken from fasted animals maintained in 5 mM glucose, ghrelin significantly inhibited the ST-EPSC amplitude in 10/14 neurons from −181 ± 12 pA to −80 ± 10 pA, an average inhibition of 54 ± 7%. Thus the average inhibition by ghrelin was significantly increased in slices prepared from fasted mice compared to slices prepared and maintained under exactly the same conditions from ad lib fed mice (; p<0.01, Student’s t-test). We did not observe any significant differences between slices from fed animals maintained in 5 mM glucose (with sucrose added to keep 301–305 mOsm) or fed animals maintained in our normal conditions of approximately 10–15mM glucose in either ST-EPSC amplitude (−237.8 ± 24 pA vs. −222 ± 33 pA) or the size of ghrelin’s inhibition (30 ± 5% vs. 26 ± 5%) (p>0.05, Student’s t-test), suggesting that altering the glucose concentration our neurons were exposed to for the duration of our experiments did not alter ghrelin’s effect. We only saw a significant difference between slices from fed vs. fasted animals (both in 5mM glucose, p<0.01, Student’s t-test). Ghrelin also increased the PPR 1.25 ± 0.04 fold in fed animals vs. 1.71 ± 0.18 fold in fasted animals in slices maintained in 5 mM glucose (p<0.05, Student’s t test). In contrast, the input resistance of the cells was not significantly different between any of the groups (Fasted, 5 mM glucose: 511 ± 72 mΩ, Fed, 5mM glucose: 675 ± 88 mΩ vs. Fed, normal: 569 ± 91 mΩ).
Figure 6 Fasting increased the size of ghrelin’s inhibition of ST-EPSCs in TH-EGFP neurons. A. Representative traces of two ST-stimulated EPSCs from non-fasted and fasted (18 hours) mice. All mice had ad lib access to water. All recordings were made under (more ...)