Abstract
Fasting and food restriction alter the activity of the mesolimbic dopamine system to affect multiple reward-related behaviors. Food restriction decreases baseline dopamine levels in efferent target sites and enhances dopamine release in response to rewards such as food and drugs. In addition to releasing dopamine from axon terminals, dopamine neurons in the ventral tegmental area (VTA) also release dopamine from their soma and dendrites, and this somatodendritic dopamine release acts as an autoinhibitory signal to inhibit neighboring VTA dopamine neurons. It is unknown whether acute fasting also affects dopamine release, including the local inhibitory somatodendritic dopamine release in the VTA. In these studies, I have tested whether fasting affects the inhibitory somatodendritic dopamine release within the VTA by examining whether an acute 24-h fast affects the inhibitory postsynaptic current mediated by evoked somatodendritic dopamine release (D2R IPSC). Fasting increased the contribution of the first action potential to the overall D2R IPSC and increased the ratio of repeated D2R IPSCs evoked at short intervals. Fasting also reduced the effect of forskolin on the D2R IPSC and led to a significantly bigger decrease in the D2R IPSC in low extracellular calcium. Finally, fasting resulted in an increase in the D2R IPSCs when a more physiologically relevant train of D2R IPSCs was used. Taken together, these results indicate that fasting caused a change in the properties of somatodendritic dopamine release, possibly by increasing dopamine release, and that this increased release can be sustained under conditions where dopamine neurons are highly active.
Keywords: fasting, dopamine, VTA, IPSC
changes in feeding affect multiple physiological systems to cause a wide range of effects. This is most evident with the development of obesity and its associated disorders that results from increased food intake. However, decreased food intake, through acute fasting and/or chronic food restriction, also affects multiple systems and behaviors. Individuals undergo fasting and/or food restriction for a variety of reasons, such as religious rituals and health and aesthetic reasons (e.g., “dieting”), but fasting/food restriction can also be due to involuntary causes such as poverty and the inability to acquire food. Although acute fasting and food restriction can have beneficial effects on some physiological systems and behaviors (Longo and Mattson 2014), they may also have negative consequences, such as the food restriction-induced increases in drug abuse and drug-related behaviors observed in animal models (see Carr 2007) and in humans (Alsene et al. 2003; Kendzor et al. 2008; Leeman et al. 2010).
Dopamine is a key neurotransmitter involved in multiple behaviors, including feeding, and the activity of dopamine circuits are strongly influenced by decreases in feeding and body weight. Chronic food restriction and its resulting weight loss causes decreased basal dopamine levels in efferent target regions, and dopamine release elicited by refeeding or drug administration is increased in chronically food-restricted animals (Carr 2007; Heffner et al. 1980; Hernandez and Hoebel 1988; Pothos et al. 1995a,b; Wilson et al. 1995). Chronic food restriction also causes cellular and molecular changes within dopamine pathways, such as increased tyrosine hydroxylase levels (Lindblom et al. 2006; Pan et al. 2006) and decreased dopamine transporter levels (Zhen et al. 2006). Furthermore, chronic food restriction affects multiple aspects of reward and reinforcement, including increasing the motivation for food and drug rewards and increasing the acquisition and total amount of food and drug consumed in self-administration assays (Bell et al. 1997; Carr 2007; Fulton et al. 2000; Jewett et al. 1995). Thus chronic food restriction strongly influences both the activity of dopamine circuits and multiple behaviors controlled by dopamine.
Although we have a good understanding of the effects of chronic food restriction on dopamine activity, much less is known about how shorter term changes in food intake, such as acute fasting, affect dopamine circuits. Acute fasting has been shown to decrease dopamine transporter levels (Patterson et al. 1998) similar to what was observed with chronic food restriction (Zhen et al. 2006). In addition, acute fasting (e.g., 24–36 h of no food) and acute food restriction (e.g., 20 h with a limited amount of food) increased motivation and responding for food and drugs in self-administration assays (Carroll 1985; Carroll et al. 1979; Jewett et al. 1995), although the increase in food responding was lower in acutely fasted rats compared with chronically food-restricted rats (Jewett et al. 1995). Thus although acute fasting is common in humans (Longo and Mattson 2014), much less is known about the overall effects of fasting on dopamine systems and dopamine-dependent behaviors, including whether fasting changes baseline dopamine levels and reward-driven dopamine release similar to chronic food restriction.
In addition to releasing dopamine from their axons at efferent target sites, dopamine neurons in the ventral tegmental area (VTA) also release dopamine locally from their soma and dendrites. This local, somatodendritic dopamine inhibits dopamine neuron activity by binding to dopamine D2 receptors (D2R) and activating G protein-coupled inwardly rectifying potassium channels (Beckstead et al. 2004; Lacey et al. 1987; Mercuri et al. 1997). D2Rs that respond to somatodendritic dopamine release in the VTA play an important role in dopamine neuron function and influence numerous behaviors mediated by dopamine (Ford 2014). For example, injection of D2R agonists into the VTA decreases basal dopamine levels, inhibits food intake, stimulates conditioned-place aversion, and reduces reinstatement of cocaine self-administration (Liu et al. 2008; Xue et al. 2011). Recently, it has also been demonstrated that selective deletion of D2R autoreceptors in dopamine neurons (without affecting postsynaptic heteroreceptor D2Rs) increased dopamine release in the striatum and affected multiple aspects of the behavioral response to cocaine, including increased locomotor activity, conditioned-place preference, and self-administration (Anzalone et al. 2012; Bello et al. 2011). D2R autoreceptors in the VTA have also recently been shown to undergo plastic changes after drug exposure (Henry et al. 1989; Madhavan et al. 2013; Marinelli et al. 2003; Perra et al. 2011; Wolf et al. 1993). Thus somatodendritic dopamine release and D2Rs in the VTA clearly play an important role in controlling dopamine neuron activity under both normal and pathological conditions. There has been little examination of the similarities and differences between the efferent axonal release of dopamine and the local somatodendritic dopamine release, however, so it is unknown whether dopamine release in these different regions are regulated by the same mechanisms. Furthermore, it is unclear whether treatments that change efferent axonal dopamine release also cause similar changes in local somatodendritic dopamine release.
Although food restriction has been shown to alter dopamine levels in efferent target regions, it is unknown whether acute fasting also affects dopamine output. In addition, it is unknown whether the local somatodendritic dopamine release within the VTA is affected by fasting or food restriction. In these studies, I have tested whether acute fasting alters somatodendritic dopamine release in the VTA by examining the effects of an acute 24-h fast on the D2R-mediated inhibitory postsynaptic current (D2R IPSC-an inhibitory postsynaptic current activated by somatodendritic dopamine release; Beckstead et al. 2004).
MATERIALS AND METHODS
Animals.
Young adult, male C57Bl/6J mice (6–11 wk old) purchased from The Jackson Laboratory (Bar Harbor, ME) were used for all experiments. All protocols and procedures were approved by the Institutional Animal Care and Use Committee at Georgia State University and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Acute fasting.
Mice were group housed (2–5 mice/cage) and given ad libitum access to food and H2O throughout the study. For acute fasting, individual mice were transferred to a new cage with no food but normal access to H2O ∼24 h before electrophysiology recordings. To control for the potential effects of social isolation during single-housing, a subset of mice were individually transferred to a new cage with normal ad libitum access to food and H2O ∼24 h before electrophysiology recordings. There were no differences between the fed, group-housed mice and the fed, single-housed mice, so data from these two groups were combined for all analyses.
Slice preparation and electrophysiology.
Mice were anesthetized with Isoflurane followed by decapitation and removal of the entire brain. The brain was removed to ice-cold, carbogen (95% O2-5% CO2)-saturated artificial cerebral spinal fluid (aCSF) and a brain block containing the VTA was prepared. The aCSF contained the following (in mM): 126 NaCl, 2.5 KCl, 2.4 CaCl2, 1.2 NaH2PO4 H20, 1.2 MgCl2, 21.4 NaHCO3, and 11.1 glucose. Pseudohorizontal slices (220 μm) containing the VTA were cut and incubated with aCSF containing 10 μM MK801 {(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate} at 35°C for ∼30 min before transfer to the perfusion chamber for electrophysiology recordings. The brain slices were constantly perfused with oxygen-saturated aCSF heated to ∼35°C at a rate of ∼1.7–2.0 ml/min. Dopamine neurons were visualized using gradient-contrast optics and were identified by their location relative to the medial terminal nucleus of the accessory optic tract (MT). Only neurons in the VTA located medial to the MT were used in these studies. Dopamine neurons were identified physiologically by the presence of spontaneous pacemaker firing (1–5 Hz), the presence of hyperpolarization-activated cation currents, and sensitivity to dopamine. Electrophysiology recordings were made using a potassium gluconate based internal solution containing the following (in mM): 128 K gluconate, 10 HEPES, 10 NaCl, 1 MgCl2, 10 BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid], 2 (Mg)ATP, 0.3 (Na)GTP, and 10 creatine phosphate. Electrodes had resistances of ∼2.3–3.0 MΩ when filled with the K gluconate internal solution. All recordings were made in voltage clamp at a holding potential of −60 mV. Series resistance values were ∼5–10 MΩ, and cells were excluded if the series resistance increased by >10% during the course of the recording.
The D2R IPSC was evoked by providing one to five stimuli (∼200 μA) at 40 Hz with a monopolar stimulating electrode located 100–300 μm posterior to the recorded cell. D2R IPSCs evoked by five stimuli were used as the baseline IPSC for these studies, as this pattern of activity mimics the phasic burst pattern of dopamine neuron firing in vivo (Beckstead et al. 2004; Grace et al. 2007). The D2R IPSC was isolated pharmacologically by including antagonists to AMPA receptors (DNQX; 10 μM), GABA-A receptors (picrotoxin; 100 μM), and GABA-B receptors (CGP55845; 0.5 μM) in the bath solution. Sulpiride (200 nM) was used at the end of each experiment to confirm that the D2R IPSC was due to dopamine release acting on D2Rs. D2R IPSCs were also evoked through the iontophoretic application of dopamine. A high resistance pipette (∼80–100 MΩ) filled with 1 M dopamine was placed in close proximity (∼10–20 μm) to the recorded cell, and dopamine was ejected as a cation with a single pulse (10 nA, 25 ms). Leak of dopamine from the iontophoretic pipette was prevented with the constant application of a small negative backing current (2.0 nA). The data were sampled at 10 kHz and filtered at 2.6 kHz using Axograph X software and an Axon MultiClamp 700B amplifier. The total current carried by the D2R IPSCs in the experiments examining the train of IPSCs (see Fig. 5) was calculated by measuring the area under the current traces from the initiation of the first IPSC to the same point in time following the decay of the final five stimulus IPSC. For the experiments utilizing aCSF with reduced extracellular calcium, the reduction in calcium was balanced by increasing the magnesium concentration to maintain a constant level of divalent ions (low Ca2+ aCSF: CaCl2 = 1.0 mM; MgCl2 = 2.6 mM).
Fig. 5.
Amplitudes of physiologically relevant trains of D2R IPSCs are increased in fasted mice. A: samples traces of five 1-stimulus IPSCs followed by a 5-stimulus IPSC at 1 Hz. B: traces in A showing the reduction in the 5-stimulus IPSC after the 1-stimulus IPSC train compared with the 5-stimulus IPSC without the preceding 1-stimulus IPSCs. C: amplitude of each IPSC in the train normalized to the first 1-stimulus IPSC. D: mean total currents carried by the 1-stimulus IPSCs (left) and all IPSCs (right) in the train. E: decrease in the amplitude of the 5-stimulus IPSC compared with a 5-stimulus IPSC without the preceding train of 1-stimulus IPSCs. Fed: n = 11; fasted: n = 8. Scale bars = 20 pA/1 s. All unpaired t-tests or Mann-Whitney rank sum tests: *P < 0.05, **P < 0.01.
Data analysis.
The electrophysiology data were analyzed using Axograph X and Microsoft Excel and were plotted using Igor Pro (Wavemetrics, Lake Oswego, OR). Statistical analysis was performed using SigmaPlot (v11.0; Systat Software). All data were initially tested for normality using a Shapiro-Wilk test and were then analyzed with an ANOVA, t-test, or Mann-Whitney ranked sum test as appropriate, with a significance level of P < 0.05 set a priori.
RESULTS
I tested whether somatodendritic dopamine release in the VTA was affected by fasting, by examining the effects of an acute, 24-h fast on the D2R IPSC (the D2R-mediated inhibitory postsynaptic currents activated by somatodendritic release of dopamine; Beckstead et al. 2004). In these studies I primarily examined D2R IPSCs evoked by five stimuli, as this pattern of activity mimics the phasic burst pattern of dopamine neuron firing in vivo (Beckstead et al. 2004; Grace et al. 2007). Examination of the D2R IPSCs in cells from fasted and fed mice indicated that there were no differences in the baseline characteristics of D2R IPSCs between fasted and fed mice (mean amplitude: fed = 84.43 ± 15.35 pA, n = 16; fasted = 82.69 ± 17.26 pA, n = 9; time to peak: fed = 0.600 ± 0.022 s, n = 14; fasted = 0.577 ± 0.023 s, n = 7). To further examine the baseline D2R IPSC in fasted and fed mice, I examined the contribution of each action potential/stimulus within the five-stimulus “burst” to the overall D2R IPSC by testing whether D2R ISPCs evoked by varying numbers of stimuli showed differences in fasted vs. fed mice. When the amplitudes of the D2R IPSCs evoked with different numbers of stimuli were normalized to the five-stimulus IPSC from the same cell for comparison, the D2R IPSC evoked by a single stimulus was significantly larger in fasted mice, with a nonsignificant trend toward an increase in the size of the D2R IPSC evoked by two stimuli as well (Fig. 1), indicating that the initial action potentials within the “burst” provide a significantly larger contribution to the D2R IPSC in fasted mice.
Fig. 1.
Fasting significantly increased the amplitude of dopamine D2 receptor (D2R) inhibitory postsynaptic currents (IPSCs) evoked with low numbers of stimuli. A: sample traces of D2R IPSCs evoked by varying numbers of stimuli in the same cell from either fed or fasted mice. B: combination of sample traces of all D2R IPSCs shown in A. C: mean amplitudes of D2R IPSCs evoked by varying numbers of stimuli (normalized to the amplitude of the 5-stimulus IPSC). Two-way repeated measures ANOVA: significant main effect of stimulus number [F(3,55) = 535.028, P < 0.001], a nonsignificant main effect of feeding status [F(1,55) = 3.655, P < 0.10], and a significant feeding status × stimulus number interaction [F(3,55) = 3.514, P < 0.05]. Fed: n = 16; fasted: n = 9. Scale bars = 20 pA/1 s. *P < 0.05.
I next examined this difference in more detail by comparing the D2R IPSCs that would be expected to occur if the five-stimulus D2R IPSC were a result of perfect temporal summation of five individual one-stimulus D2R IPSCs. As has been shown previously (Beckstead et al. 2007), the scaled arithmetic sum of five one-stimulus D2R IPSCs (calculated by multiplying individual one-stimulus D2R IPSCs by 5) is larger than the observed five-stimulus D2R IPSC (Fig. 2). Comparison of the expected arithmetic sum D2R IPSCs from fed and fasted mice demonstrated that the summed D2R IPSC from fasted mice was significantly increased compared with fed mice (Fig. 2), confirming that the first stimulus provided a significantly greater contribution to the overall D2R IPSC in fasted mice.
Fig. 2.
Fasting increases the contribution of the first stimulus in the D2R IPSC. A and B: comparison of the 1-stimulus D2R IPSC, the 5-stimulus IPSC, and the arithmetic sum of 5 1-stimulus D2R IPSCs showing perfect temporal summation from fed (A) and fasted (B) mice. C: comparison of the arithmetic sum of five 1-stimulus D2R IPSCs from fed and fasted mice. Unpaired t-tests: *P < 0.05, #P < 0.09. D: mean D2R IPSC amplitudes (normalized to the 5-stimulus D2R IPSC) of the 1-stimulus and 5 × 1-stimulus arithmetic sum D2R IPSCs. Fed: n = 16; fasted: n = 9. Unpaired t-tests: *P < 0.05.
I next evoked two consecutive D2R IPSCs to further examine potential fasting induced changes in the D2R IPSC. D2R IPSCs evoked by five stimuli were induced either 2 or 5 s apart, and the magnitude of the decrease in the amplitude of the second D2R IPSC was examined by calculating the ratio of the amplitudes of the two D2R IPSCs (D2R IPSC ratio: IPSC2/IPSC1). In addition, because the first stimulus provided a significantly larger contribution to the D2R IPSC in fasted mice, I also examined the ratios of repeated one-stimulus D2R IPSCs. For both the five-stimulus IPSCs and the one-stimulus IPSCs, the D2R IPSC ratios were significantly larger in fasted animals when the IPSCs were separated by only 2 s (Fig. 3, A and B), indicating that the second evoked D2R IPSC was significantly larger in dopamine neurons from fasted mice. There were no differences in the ratios of five-stimulus or one-stimulus IPSCs when the pulses were separated by a longer, 5-s interval, however, (Fig. 3, A and B).
Fig. 3.
Fasting increases the ratio of the amplitudes of repeated D2R IPSCs at short intervals but not longer intervals. A: samples traces of repeated D2R IPSCs evoked with 5 stimuli (left) or 1 stimulus (right) and separated by 2 (top) or 5 s (bottom). B: mean D2R IPSC ratios (IPSC2/IPSC1) of 1-stimulus and 5-stimulus IPSCs separated by 2 and 5 s. Unpaired t-tests, fed vs. fasted: *P < 0.05. C: sample traces of a 5-stimulus IPSC alone and 2 s after a 1-stimulus IPSC. D: mean %decrease in the amplitude of the 5-stimulus IPSC 2 s after a 1-stimulus IPSC (left) and mean IPSC ratios of the 5 stimulus IPSC compared with the 1-stimulus IPSC (IPSC5STIM/IPSC1STIM) (right). Unpaired t-tests or Mann-Whitney rank sum test: P > 0.05. Fed: n = 9–14; fasted: n = 7–8. Scale bars = 20 pA/1 s for all 5-stimulus IPSCs and 10 pA/1 s for the 1 stimulus IPSCs in A.
Because of the increased contribution of the first stimulus to the D2R IPSC and the increased D2R IPSC ratios for both one-stimulus and five-stimulus D2R IPSCs, I also examined whether evoking a one-stimulus IPSC before a five-stimulus IPSC showed similar differences in the D2R IPSC ratios in fasted vs. fed mice. Interestingly, fasting had no effect on the D2R IPSC when a one-stimulus IPSC was evoked 2 s before a five-stimulus IPSC (Fig. 3C-D). The ratios of the D2R IPSCs were not affected, and the addition of the one-stimulus IPSC reduced the amplitude of the five-stimulus IPSC to a similar degree in both fasted and fed mice (compared to a five-stimulus IPSC evoked alone; Fig. 3, C and D).
I next examined whether the calcium sensitivity of dopamine release was also affected by fasting. As has been shown previously (Courtney and Ford 2014), reducing extracellular calcium in the aCSF from 2.4 to 1.0 mM significantly reduced the amplitude of both the five-stimulus D2R IPSC (Fig. 4) and the one-stimulus D2R IPSC without changing the D2R IPSC ratios in either fed or fasted mice (Fig. 4C). Reducing extracellular calcium did have a significantly larger effect on the amplitudes of the D2R IPSCs from fasted mice however, (Fig. 4, B, D, and E). Similar results were observed when examining one-stimulus D2R IPSCs (%decrease: IPSC1: fed = 43.9 ± 5.1%, fasted = 54.9 ± 4.4%; IPSC2: fed = 30.3 ± 9.1%, fasted = 38.2 ± 5.2%; fed: n = 4, fasted: n = 3), but due to the small size of these D2R IPSCs and the magnitude of the decrease when switching to low calcium aCSF, it was difficult to accurately quantify the effect of low calcium on the one-stimulus D2R IPSC.
Fig. 4.
D2R IPSC from fasted mice is more sensitive to reduced extracellular calcium. A: sample traces of D2R IPSCs (5-stimulus IPSCs; 2-s interval) from fed and fasted mice in normal (black trace) and low calcium (1.0 mM; gray traces) artificial cerebral spinal fluid (aCSF). B: decrease in peak amplitude of the D2R IPSC caused by low calcium aCSF. Unpaired t-tests: *P < 0.05, #P < 0.10. C: changes in the D2R IPSC ratios caused by low calcium aCSF. Unpaired t-tests: P > 0.05. D and E: time course of the reduction in the D2R IPSC in low calcium aCSF for IPSC#1 (D) and IPSC#2 (E). Arrows in D and E indicate time of switch to low calcium aCSF. IPSC#1: significant main effects of feeding status F(1,223) = 7.205, P = 0.018, and time F(16,223) = 35.37, P < 0.001; and a significant feeding status × time interaction F(16,223) = 2.172, P = 0.007 (*P < 0.05, #P < 0.1); IPSC#2: no significant main effect of feeding status (P = 0.228) and no significant feeding status × time interaction (P = 0.136). Scale bars = 20 pA/1 s; n = 8–10.
I next sought to compare D2R IPSCs from fasted and fed mice using a more physiological stimulus protocol that mimics dopamine neuron firing in vivo, where bursts of action potentials follow trains of tonic, pacemaker action potential firing (Grace et al. 2007). To test this firing pattern, a five-stimulus D2R IPSC was evoked after a train of five one-stimulus D2R IPSCs stimulated at 1 Hz. As expected, the amplitude of the one-stimulus IPSCs decreased with each subsequent IPSC, and the amplitude of the five-stimulus IPSC was significantly reduced compared with a five-stimulus IPSC evoked alone in the same cell (Fig. 5, A–C, and E). The amplitudes of both the five-stimulus IPSC and the one-stimulus IPSCs preceding it were significantly larger in dopamine neurons from fasted mice when normalized to the initial one-stimulus IPSC (Fig. 5C), as would be expected based on the increased D2R IPSC ratios observed above (see Fig. 3). In addition, fasted mice had a significantly smaller reduction in the amplitude of the five-stimulus D2R IPSC caused by the preceding one-stimulus train (compared to a control, single five-stimulus IPSC from the same cell; Fig. 5, B and E). Finally, the total current carried by both the five one-stimulus IPSCs and all six IPSCs in the train as significantly larger in fasted mice compared with fed mice (Fig. 5D). Thus, overall, both the train of “pacemaker” one-stimulus D2R IPSCs and the “burst” five-stimulus D2R IPSC following the train were significantly larger in fasted mice.
I next tested whether forskolin and l-DOPA, which use distinct methods to increase the amplitude of the D2R IPSC without affecting the ratios of repeated D2R IPSCs (Beckstead et al. 2007), have similar effects on D2R IPSCs from fasted and fed mice. Forskolin (5 μM), which increases the probability of neurotransmitter release at many synapses, increased the amplitude of both D2R IPSCs equally, without affecting the D2R IPSC ratio in either fed or fasted mice (Fig. 6, A and C). The magnitude of the forskolin-induced increase in the amplitude of the D2R IPSCs was significantly reduced in cells from fasted mice, however, (Fig. 6, A, B, D, and E). Similarly, l-DOPA (10 μM), which increases the amount of dopamine loaded per vesicle, increased the amplitude of both D2R IPSCs without altering the D2R IPSC ratios in both fed and fasted mice (Fig. 6, F and H). Unlike forskolin however, l-DOPA had a similar effect on the amplitude of the D2R IPSCs in fasted and fed mice (Fig. 6, F, G, I, and J). Thus the effects of forskolin, but not l-DOPA, were significantly reduced by fasting.
Fig. 6.
Fasting reduces the increase in the D2R IPSC caused by forskolin but not l-DOPA. A and F: sample traces; B and G: %increases in peak amplitude (Mann-Whitney rank sum tests: *P < 0.05); C and H: changes in D2R IPSC ratios (unpaired t-tests: P > 0.05); D, E, I, and J: time courses of the change in amplitude of D2R IPSCs from fed and fasted mice after forskolin (A–E; 5 μM) or l-DOPA (F–J; 10 μM). Bars in D, E, I, and J indicate time of forskolin (D and E) or l-DOPA (I and J) application. D: significant feeding status × time interaction, F(15,207) = 2.336, P = 0.004 (*P < 0.05, #P < 0.1), but no significant main effect of feeding status F(1,207) = 3.487, P = 0.083. E: significant feeding status × time interaction, F(15,204) = 2.945, P < 0.001 (*P < 0.05, #P < 0.1), but no significant main effect of feeding status F(1,204) = 3.251, P = 0.093. I and J: no significant main effect of feeding status and no significant feeding status × time interaction (P > 0.05). Forskolin: fed n = 8; fasted n = 7. l-DOPA: fed n = 8; fasted n = 8. Scale bars = 20 pA/1 s.
Although these data indicate that fasting affects the presynaptic release of dopamine, it is possible that alterations in postsynaptic D2R signaling could have also influenced the fasting-induced changes observed above. Thus I next tested whether acute fasting affected the desensitization of postsynaptic D2Rs in VTA dopamine neurons. We initially tested D2R signaling and desensitization through the use of the iontophoretic application of dopamine to isolate postsynaptic D2R signaling and to eliminate any potential effects of changes in presynaptic dopamine release. Two iontophoretic D2R IPSCs were evoked 2 or 5 s apart and the ratio of the IPSCs was calculated. Unlike for electrically evoked D2R IPSCs, the second iontophoretic D2R IPSC was not decreased in either fed or fasted mice relative to the first IPSC at either time interval (Fig. 7, A and B), and there even appeared to be a slight facilitation of the second D2R IPSC with the 2-s interval. This indicates that there is no desensitization of the D2Rs when D2R IPSCs are evoked close together and that changes in postsynaptic D2Rs are not likely to contribute to the fasting-induced changes observed above. As a further test of potential changes in postsynaptic D2Rs, I also examined whether fasting changed the acute desensitization of D2Rs in response to bath application of a maximal dose of the D2R agonist quinpirole (3 μM) (Perra et al. 2011). Although there appeared to be a slight increase in D2R desensitization in dopamine neurons from fasted mice, overall there were no significant differences in the timing or magnitude of D2R desensitization between fasted and fed mice (Fig. 7, C–E). As a final test for potential changes in postsynaptic D2Rs, I also performed a dose-response curve for the inhibition of the iontophoretic D2R IPSC by the D2R antagonist sulpiride. Sulpiride inhibited the D2R IPSC by the same amount in fed and fasted mice (Fig. 7F) with no difference in the calculated IC50 values (fed: IC50 = 1.66 pM; fasted: IC50 = 1.66 pM). Thus there do not appear to be any changes in postsynaptic D2Rs in acutely fasted mice.
Fig. 7.
Postsynaptic D2Rs are not affected by fasting. A: sample traces of repeated D2R IPSCs elicited by iontophoretic application of dopamine and separated by 2 s (top) or 5 s (bottom). B: mean D2R IPSC ratios (IPSC2/IPSC1) of iontophoretic D2R ISPCs separated by 2 and 5 s. Unpaired t-tests: P > 0.05. C: sample traces of quinpirole (3 μM)-induced currents from fed and fasted mice. D: average quinpirole-induced currents from fed and fasted mice (normalized to the peak quinpirole-induced current). Two-way repeated-measures ANOVA: no significant main effects of feeding status, and no significant feeding status × time interaction; P > 0.05. E: mean desensitization of quinpirole-induced currents. Unpaired t-tests: P > 0.05. F: dose-response curve of inhibition of iontophoretic D2R IPSCs by sulpiride. Black bars in C and D indicate time of quinpirole (3 μM) application and grey bars indicate time of Sulpiride (200 nM) application. Fed: n = 6–12; fasted: n = 4–9. Scale bars = 20 pA/1 s (A) and 50 pA/1 s (C and D).
DISCUSSION
In these studies I have tested whether acute fasting affects the local somatodendritic release of dopamine within the VTA by testing the effects of an acute 24-h fast on the evoked D2R IPSC. Overall, these studies indicate that acute fasting altered the properties of somatodendritic dopamine release within the VTA, possibly by increasing dopamine release. Fasted mice had larger contributions of the first stimulus/action potential to the overall D2R IPSC (Figs. 1–2), an increase in the D2R IPSC ratios when repeated IPSCs were evoked at short intervals (indicating an increase in the amplitude of the second D2R IPSC) regardless of number of stimuli used to evoke the D2R IPSC (Fig. 3), significantly reduced release in low calcium aCSF (Fig. 4), a reduced response to forskolin (Fig. 6), and bigger ISPCs when a more physiologically relevant train of IPSCs was used (Figure 5). Taken together, these results suggest that fasting caused an increase in somatodendritic dopamine release, and that this increased release can be sustained under conditions where dopamine neurons are highly active.
An increase in the probability of somatodendritic dopamine release could explain both the increase in the D2R IPSC evoked by low numbers of stimuli (Figs. 1–2), and the significantly larger effect that reducing extracellular calcium had on the D2R IPSC (Fig. 4). In addition, the decrease in the amplitude of the second D2R IPSC when stimulating repeated IPSCs has previously been shown to be mediated by a presynaptic mechanism (Beckstead et al. 2007), further supporting the hypothesis that the increased ratios of repeated D2R IPSCs (IPSC2/IPSC1) in fasted mice (Fig. 3) were due to a sustained increase in presynaptic dopamine release. A fasting-induced increase in dopamine release is also supported by the effects of forskolin on the D2R IPSC, as the ability of forskolin to increase the D2R IPSC was significantly reduced in fasted mice (Fig. 6). Forskolin is thought to increase synaptic transmission by increasing the probability of neurotransmitter release, and I have shown previously that forskolin increases the D2R IPSC solely via a presynaptic mechanism, as it does not affect the postsynaptic response to iontophoretically applied dopamine (Roseberry et al. 2007). Thus, if dopamine neurons from fasted mice indeed do have a higher probability for dopamine release, the effects of forskolin should be reduced in fasted mice, which is what was observed in these studies.
Paired-pulse stimulation, forskolin, and reduced extracellular calcium have all been widely used to examine neurotransmitter release in different regions of the brain. Typically the studies using these approaches have examined the release of fast-acting (i.e., millisecond time scale) neurotransmitters that mediate their effects through activation of ionotropic receptors (i.e., GABAA, AMPA) however, whereas in these studies I have utilized these approaches to examine postsynaptic responses mediated by the activation of G-protein coupled receptors. As these D2R-mediated responses are much slower than the fast ionotropic responses and are dependent on the activation of intracellular signaling, the interpretation of changes in the response to forskolin, reduced extracellular calcium, and paired-pulse stimulation is more complicated. The repeated D2R IPSCs evoked in these studies are not traditional paired-pulse experiments as they are normally used for ionotropic receptor mediated responses. The repeated D2R IPSCs were separated by 2–5 s in these experiments compared with the millisecond-level timing of traditional paired-pulse depression. In addition, trains of stimuli were used to mimic the in vivo pattern of dopamine neuron burst firing in these studies, which leads to multiple individual action potential driven dopamine release events that collectively converge on the postsynaptic D2Rs to give a single postsynaptic response. Therefore, the repeated D2R IPSCs are most likely not measuring dopamine release probability, but the ability to maintain sustained release in response to repeated dopamine burst firing. Thus, although the results of the experiments utilizing repeated D2R IPSCs, forskolin, and reduced extracellular calcium indicate that fasting increases presynaptic dopamine release, likely through an increase in the probability of release, it is unknown if the mechanisms underlying the responses to these treatments are the same for the D2R-mediated responses measured here compared with the widely examined fast ionotropic receptor-mediated responses typically studied.
It is possible that decreases in the desensitization of D2Rs could have contributed to these effects, especially for the increase in D2R IPSC ratios of D2R IPSCs evoked at short intervals. However, when repeated D2R IPSCs were evoked by the iontophoretic application of dopamine, the amplitude of both D2R IPSCs was the same in both fed and fasted mice. In addition, there was no difference between fed and fasted mice in the rate or extent of desensitization in response to the bath application of the D2R agonist quinpirole (3 μM). These findings strongly suggest that decreased desensitization of D2Rs did not contribute to the increases in the D2R IPSC observed in these studies. Although there appeared to be a slight increase in D2R desensitization in fasted mice in response to quinpirole, the overall lack of an effect of acute fasting on D2R desensitization is in contrast with a recent article (Branch et al. 2013) that showed that prolonged food restriction increased desensitization of D2Rs in VTA dopamine neurons. Although this suggests that there may be differences in D2R desensitization between acute fasting and more prolonged food restriction, it is also possible that the different experimental approaches used in these studies may be the cause of the observed differences. For example, Branch et al. (2013) used prolonged iontophoretic application of dopamine to examine desensitization compared with the acute iontophoresis and bath application of the D2R agonist quinpirole used in this study. In addition, the differences in calcium buffering of the intracellular solutions between these studies may also have contributed to the observed differences in desensitization. It has previously been shown that D2R desensitization is particularly sensitive to intracellular calcium levels (Beckstead and Williams 2007). As Branch et al. (2013) used a low calcium buffering internal solution (0.4 mM EGTA) compared with the high calcium buffering solution used here (10 mM BAPTA), the difference in intracellular calcium levels may underlie the differences in desensitization observed in these two studies. Despite these differences and their underlying cause, it does not appear that altered desensitization of D2Rs contributed to the changes observed in the studies presented here.
Changes in feeding have been widely shown to alter the activity of dopamine circuits and to affect multiple dopamine-dependent behaviors. As described above, food restriction decreases baseline dopamine levels, increases dopamine output in response to rewards, and increases multiple reward-related behaviors (Bell et al. 1997; Carr 2007; Carroll 1985; Carroll et al. 1979; Fulton et al. 2000; Heffner et al. 1980; Hernandez and Hoebel 1988; Jewett et al. 1995; Lindblom et al. 2006; Pan et al. 2006; Patterson et al. 1998; Pothos et al. 1995a,b; Wilson et al. 1995; Zhen et al. 2006). Interestingly, increased food intake and the development of obesity also change the activity of dopamine circuits and dopamine-dependent behaviors (Kenny 2011; Volkow et al. 2011), but in a manner opposite to the changes observed with food restriction. Whereas food restriction increases dopamine responses in efferent target regions such as the striatum (Carr 2007), obesity appears to decrease the dopamine responses to rewards (Kenny 2011; Volkow et al. 2011). For example, chronic food restriction has been shown to increase the behavioral response to D1R agonists as well as to increase D1R signaling in the ventral striatum (Carr 2007). In contrast, obesity appears to decrease dopamine responses in part though a reduction in the function of D2Rs. Rats given access to a high-fat cafeteria style diet developed obesity with a level of weight gain that was inversely proportional to the expression of D2Rs in the ventral striatum. Prolonged intake of the high-fat diet in this study also led to increased reward thresholds (indicating a decrease in reward sensitivity), and striatal knockdown of D2Rs similarly increased reward thresholds in rats given acute access to the cafeteria diet (Johnson and Kenny 2010). Furthermore, multiple human studies have demonstrated a decrease in dopamine output in the ventral striatum in response to rewarding foods in obese individuals, and striatal D2Rs levels have been shown to correlate with both obesity levels and the decreased dopamine response in obese individuals (Kenny 2011; Volkow et al. 2011). Thus feeding status has a strong influence over the function of dopamine pathways, although at this point it is unclear whether there are distinct mechanisms underlying the food restriction- and obesity-driven changes in dopamine circuits or whether there is a common mechanism operating to cause these opposite effects depending on feeding status.
Fasting causes numerous physiological changes that could potentially impact dopamine neurons to mediate the alterations in somatodendritic dopamine release observed in these studies. For example, fasting and food restriction are both stressful, and stress has profound influence on the activity of dopamine neurons and dopamine-dependent behaviors. Thus it is possible that changes in stress hormones or stress-responsive pathways could affect dopamine neurons to contribute to these changes, although further studies will be required to test the potential contribution of the multiple components of stress pathways that could contribute to these effects. Fasting also affects the activity of multiple neurotransmitters, neuropeptides, and hormones that have been shown to affect dopamine neuron activity, and further study will be required to determine whether fasting-induced changes in other neurotransmitters or neuropeptides may contribute to these effects.
Chronic food restriction decreases baseline dopamine at efferent target regions and results in enhanced dopamine release in response to rewarding and reinforcing substances such as food and drugs (Carr 2007; Heffner et al. 1980; Hernandez and Hoebel 1988; Pothos et al. 1995a,b; Wilson et al. 1995). Our results demonstrating that fasting increases somatodendritic dopamine release in the VTA agree with these previous studies, as well as the recent study showing that dopamine neurons were more excitable following chronic food restriction, with increased burst firing in vivo and increased probability of burst firing in response to glutamate in vitro (Branch et al. 2013). However, the local somatodendritic dopamine release within the VTA is inhibitory to overall dopamine neuron activity, so an increase in somatodendritic dopamine release would be expected to decrease overall dopamine neuron activity, which would contradict the increased dopamine release at efferent target sites that has been observed with chronic food restriction. One potential explanation for this apparent discrepancy is that the increased somatodendritic dopamine release in the VTA may reduce the baseline tonic activity of VTA dopamine neurons to facilitate the “phasic” burst firing in response to rewards. The observed increase in somatodendritic dopamine release in response to a single action potential (Figs. 1–3) and in response to slow trains of single action potentials (Fig. 5) could lead to an overall decrease in VTA dopamine neuron activity during the “tonic” phase of VTA dopamine neuron firing and the reduced baseline extracellular levels of dopamine in efferent target regions that has been observed following chronic food restriction (Pothos et al. 1995a,b). This decrease in tonic activity could then also facilitate the response to the “phasic” burst-induced dopamine release caused by salient stimuli that has also been observed with chronic food-restriction. In addition, the sustained increase in the somatodendritic dopamine release that was observed when evoking consecutive “burst” D2R IPSCs close together (Fig. 3) may allow for an animal to be able to respond to multiple salient stimuli presented consecutively, which could be advantageous to a fasted/food-restricted animal as it searches for food.
The local inhibition of VTA dopamine neurons caused by somatodendritic dopamine release has also been proposed to be involved in generating the pause in firing that has been observed following a burst of action potentials, and this pause may be involved in synchronizing the activity of populations of dopamine neurons to allow them to coordinately burst fire in response to salient stimuli (Beckstead et al. 2004; Schultz 1998). Thus the sustained increase in dopamine release in response to an acute fast may also increase the synchrony of VTA dopamine neurons to promote an increased response to rewards. However, further studies will be required to identify the exact role of the increased somatodendritic dopamine release caused by fasting and how it relates to changes in dopamine release at efferent terminal regions, such as the nucleus accumbens.
GRANTS
Funding for these studies was provided by the Department of Biology and the Brains and Behavior Program at Georgia State University.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: A.G.R. conception and design of research; A.G.R. performed experiments; A.G.R. analyzed data; A.G.R. interpreted results of experiments; A.G.R. prepared figures; A.G.R. drafted manuscript; A.G.R. edited and revised manuscript; A.G.R. approved final version of manuscript.
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