Pre‐ and post‐synaptic modulation by GABAB receptors of rat neuroendocrine dopamine neurones

The secretion of prolactin from the pituitary is negatively controlled by tuberoinfundibular dopamine (TIDA) neurones. The electrical properties of TIDA cells have recently been identified as a modulatory target of neurotransmitters and hormones in the lactotrophic axis. The role of the GABAB receptor in this control has received little attention, yet is of particular interest because it may act as a TIDA neurone autoreceptor. Here, this issue was explored in a spontaneously active rat TIDA in vitro slice preparation using whole‐cell recordings. Application of the GABAB receptor agonist, baclofen, dose‐dependently slowed down or abolished the network oscillations typical of this preparation. Pharmacological manipulations identify the underlying mechanism as an outward current mediated by G‐protein‐coupled inwardly rectifying K+‐like channels. In addition to this postsynaptic modulation, we describe a presynaptic modulation where GABAB receptors restrain the release of glutamate and GABA onto TIDA neurones. Our data identify both pre‐ and postsynaptic modulation of TIDA neurones by GABAB receptors that may play a role in the neuronal network control of pituitary prolactin secretion and lactation.


| INTRODUC TI ON
Neurotransmitter regulation of the neuroendocrine system has commonly been studied and discussed with regard to the role of neuropeptides and monoamines. Yet, similar to the rest of the central nervous system (CNS), hypothalamic interneuronal communication depends critically on signalling via glutamate and GABA, amino acid transmitters that are near-ubiquitous within the neuroendocrine networks, [1][2][3] and specialised GABAergic nerve endings throughout the hypothalamus were identified early. 4 Glutamate and GABA not only reach neurones through incoming projections, but also as co-transmitters within neuroendocrine neurones. One prominent example of the latter is the tuberoinfundibular dopamine (TIDA) neurones in the arcuate nucleus (ARC), which control pituitary prolactin release by exerting a tonic inhibitory influence. 5,6 This powerful inhibition constrains prolactin release to specific reproductive events such as late pregnancy, nursing and a pre-ovulatory surge in the oestrus cycle. 7 In the juvenile male rat, TIDA neurones discharge rhythmically and in synchrony, coordinated through strong electrical gap junction-mediated coupling, as revealed in in vitro studies. 8,9 Recent work has shown that the temporal properties of this oscillation are subject to ultrashort feedback loop regulation exerted by dopamine acting on autoreceptors at the somatodendritic level of TIDA neurones. 10 In addition to dopamine, TIDA neurones appear to signal via the inhibitory neurotransmitter γ-aminobutyric acid (GABA), as first suggested by the coexistence of immunoreactivity for glutamic acid decarboxylase and tyrosine hydroxylase, enzymes of the GABA and dopamine biosynthetic pathways, respectively. 11,12 The GABAergic identity of at least a subpopulation of neuroendocrine dopamine neurones is further supported by subsequent studies using ultrastructure, 13 reporter gene expression [14][15][16] and optogenetics. 17 The role of GABA in TIDA neurones is, however, poorly understood. Most studies have focused on possible actions in the pituitary, parallel to the inhibitory influence that dopamine exerts on lactotrophs. Indeed, the GABA B receptor is expressed in the anterior pituitary gland, 18 and application of GABA on isolated pituitaries attenuates prolactin release. 19,20 The possibility that GABA can act on auto-or heteroreceptors within the TIDA system to regulate the electrical activity of dopaminergic neurones has received little attention. There is good reason to believe that TIDA neurones are subject to GABAergic modulation of their membrane and network properties, which may play a role in shaping the normal rhythms of prolactin release. Indeed, both mRNA 21,22 and protein 23 for the metabotropic GABA B receptor are found in the ARC. Electrophysiological studies have also identified G protein-coupled inwardly rectifying K + (GIRK)like currents, a common target for GABA B receptor activation in TIDA neurones, 17,24,25 and some ARC neurones respond to GABA B agonists by hyperpolarisation. 26,27 Notably, stimulation of GABA B receptors causes circulating prolactin to rise. 28,29 Here, we investigated the effects of modulating the activity of the GABA B receptor on TIDA neurones aiming to provide a better understanding of the role of GABA in the lactotropic axis.

| Electrophysiology: solutions, data acquisition and analysis
Whole cell voltage clamp and current clamp recordings were obtained from TIDA neurones identified on the basis of their typical electrophysiological signature. 8  Hepes, pH 7.3, 280-290 mOsm. TIDA neurones were recorded during a control period of 10 minutes (control condition) and then tested under bath application of drug. Each slice was subjected to only one application of pharmacological agents. Mean membrane potential (or, when applicable, current) values were determined as the average over 10 seconds sampled before drug application, and at the height of effect induced by the drug. When no effect was evident, signal was sampled with a delay similar to the delay observed to obtain full TTX effect. Oscillation frequency was obtained as the inverse of the time to complete five full oscillation cycles (UP + DOWN state), divided by five. Access resistance was monitored throughout the experiments, and neurones in which the series resistance exceeded 15 MΩ or changed ≥ 20% were excluded from the statistics. Liquid junction potential was 16.4 mV and was not compensated.

| Statistical analysis
Statistical significance for all analyses was determined using a twotailed Student's t test in prism (unpaired when comparing two populations of cells and paired when comparing conditions in the same cell population). All data values are reported as the mean ± SEM unless otherwise indicated. P ˂ 0.05 was considered statistically significant.

| GABA B receptor activation dose-dependently abolishes or decreases the frequency of TIDA network oscillations
TIDA neurones can be reliably identified in in vitro rat preparations on the basis of their electrophysiological properties 8,9 including a synchronised robust membrane potential oscillation, such that TIDA cells alternate rhythmically between depolarised UP states (during which action potential discharge commonly appears) and hyperpolarised DOWN states (during which discharge is absent) Input resistance was determined in slices exposed to tetrodotoxin (TTX) (500 nmol L -1 ), a treatment that abolishes TIDA oscillations, likely as a result of the blocking actions of this drug on the persistent Na + current 33   GABA B receptors are G protein-coupled receptors that activate different downstream effectors. 34 The best characterised of these are the G protein-coupled inwardly rectifying K + (GIRK) channels. 35 that is lower than that calculated for K + . This hyperpolarised value is best explained by mediation via K + flux. 25 Next, we explored the sensitivity of the TIDA neurone I Baclofen to Ba 2+ , a wide spectrum blocker of inwardly rectifying K + channels. 37 In the presence of Ba 2+ (300 µmol L -1 ), the inward current induced by baclofen (10 µmol L -1 ) at V command −60 mV (and in the presence of TTX) was diminished by 47.4 ± 7.5% (Control: 16.8 ± 2 pA; Baclofen: 10 µmol L -1 , 9.1 ± 2 pA; n = 5 cells; P = 0.001; paired t test) (Figure 2Ba,b). Finally, we tested whether I Baclofen requires the binding of G-proteins by using the sulfhydryl alkylating agent NEM, which uncouples G-protein from receptors. 38

| Little evidence for endogenous GABA B activation in vitro
The spontaneously active TIDA preparation 8 presents an opportunity to study the role of endogenously released transmitters within the slice, as is the case for e.g. dopamine. 10 To evaluate the possibility of ongoing GABA B receptor activation resulting from GABA release from TIDA neurones and/or other cells within the slice, we applied CGP55845, a selective GABA B -type antagonist. 39   Previous work has shown that the frequency of IPSCs (but not EPSCs) differs across the TIDA oscillatory cycle, with more IPSCs during the UP phase, 8 and that dopamine autoreceptor activation differentially affects excitatory and inhibitory synaptic input over the stages of the cycle. 10 The differential sIPSC frequency was confirmed in the course of the present study (UP state sIPSC frequency: 5.6 ± 0.6 Hz; DOWN state sIPSC frequency: 3.6 ± 0.9 Hz; n = 6 cells;  3.6 ± 0.9 Hz; Baclofen: 1.9 ± 0.5 Hz; n = 6 cells; P = 0.006; paired F I G U R E 1 Application of the GABA B agonist, baclofen, slows down or abolishes tuberoinfundibular dopamine (TIDA) oscillations in a concentration-dependent manner. Aa, Current clamp recording of a TIDA neurone in vitro. Application of 10 µmol L -1 baclofen results in hyperpolarisation of TIDA membrane potential and reversible abolition of phasic discharge. Dashed line in black represents membrane potential at −60 mV in all represented recordings. Ab, Quantification of recordings as that shown in (Aa) (grey lines represent individual data points; black line represents the mean ± SEM; n = 5 cells, P = 0.002, paired t test). Ba, Application of 1 µmol L -1 baclofen during a current clamp recording of an oscillating TIDA neurone results in a complete silencing of the oscillating activity and depolarisation of TIDA membrane potential compared to the oscillation nadir. Bb, Quantification of the effect illustrated as in Ab (n = 5 cells, P = 0.002, paired t test). Ca, In the presence of 0.1 µmol L -1 baclofen, the oscillation frequency of TIDA neurones slows down (n = 7 cells, P = 0.009, paired t test), which is not associated with a change in the membrane potential of TIDA neurones. Cb, Superimposed black (control) and grey (0.1 µmol L -1 baclofen) traces at increased temporal resolution. Cc, Quantification of the effect illustrated as in Ab (n = 7 cells, P = 0.009, paired t test). Da, In the presence of 0.01 µmol L -1 baclofen, no change is observed in TIDA oscillation frequency (n = 5, P = 0.8289, paired t test) or membrane potential (n = 5 cells, P = 0.585, paired t test). Db, Superimposed black (control) and grey (0.01 µmol L -1 baclofen) traces at increased temporal resolution. Dc, Quantification of the effect illustrated as in (Ab). Ea, A TIDA neurone recorded in current clamp mode in the presence of tetrodotoxin (TTX). Application of baclofen (1 µmol L -1 ) results in a hyperpolarisation. To compare change in input resistance under identical membrane potential, negative constant current is injected (−20 pA, 500 ms). Input resistance was tested before (*) and during application of baclofen (*). Eb, Illustration of the test pulse protocol and the voltage response (Control: black trace; baclofen: grey trace). Ec, Application of baclofen results in a decrease of input resistance (From 1547 ± 76 MΩ to 1376 ± 69 MΩ) (n = 5 cells, P = 0.011, paired t test). Ed, Quantification of the baclofen-induced hyperpolarisation in the presence of TTX. *P ˂ 0.05, **P ˂ 0.01, ns, not significant F I G U R E 2 Stimulation of GABA B receptors on tuberoinfundibular dopamine (TIDA) neurones activates G protein-coupled inwardly rectifying K + (GIRK)-like channels. Aa, Voltage clamp recording of a TIDA neurone in vitro clamped at −60 mV. After application of TTX (500 nmol L -1 ; resulting in a cessation of oscillations), subsequent application of baclofen (10 µmol L -1 ) yields a reversible outward current (I Baclofen ). Dashed line in red represents 0 pA holding current. Ab, Mean peak current amplitude in control and in the presence of baclofen (grey lines represent individual data points; black line represents the mean ± SEM). Ac, Averaged holding current frequency distribution from recordings as that illustrated in Aa; in control (black) and in the presence of baclofen (red; n = 11 cells). Ad, Voltage clamp ramp protocol to extract I Baclofen over a spectrum of membrane potentials. Ae, Averaged baclofen induced current obtained by the digital subtraction of voltage-clamp ramps performed in control and at peak of baclofen response (n = 5 cells). The estimated reversal potential (E Rev ) of the I Baclofen is −123.

| D ISCUSS I ON
The pharmacological control of the neuroendocrine system has primarily been studied in the context of neuropeptidergic and (This value is more hyperpolarised than the calculated reversal potential for K + at −111 mV, 25 although it may be explained by the presence of strong gap junction coupling in the rat TIDA network, 9 which negatively impacts on space clamp.) The partial sensitivity of I Baclofen to Ba + suggests that it corresponds to an inwardly rectifying K + current 37 (but that additional, as yet unidentified, currents may also contribute). The failure of baclofen to elicit an outward current in the presence of NEM indicates that I Baclofen is dependent on intact G-protein signalling. 46 Combined, these findings offer strong evidence that GABA B receptors activate a GIRK current in TIDA neurones, as has been shown elsewhere in the CNS. Indeed, GIRK channels have been demonstrated as targets of μ-opioid receptors 17,24 and serotonin (via the 5-HT 1A receptor; 25 ) on TIDA neurones. Earlier work has also reported the presence of GIRK channels and baclofen-mediated hyperpolarisation in the arcuate nucleus. 26,27 Thus, GIRK channels may form a pharmacological target on TIDA cells that integrate the actions of several neuromodulators in the control of prolactin secretion. It should be noted, however, that there may be divergence of signalling pathways that rely on GIRK ac- In addition to the postsynaptic effects, we also found that a decrease in the frequency of both excitatory and inhibitory synaptic input to TIDA neurones in the presence of baclofen. The role of presynaptic GABA B receptors regulating both excitatory and inhibitory impulse traffic is well documented in other CNS regions. 32,62 Thus, the overall effect of baclofen on TIDA network activity may also include actions at the terminal level. In the intact brain, postsynaptic (somatodendritic) and presynaptic (terminal) GABA B receptors could be activated in isolation (in contrast to the in vitro F I G U R E 4 Attenuation of spontaneous inhibitory and excitatory post-synaptic currents by GABA B receptor activation. Aa, Voltage clamp recording of a tuberoinfundibular dopamine (TIDA) neurone in vitro, clamped at −80 mV. Recordings of spontaneous inhibitory post-synaptic currents (sIPSCs) were performed while blocking glutamatergic synaptic transmission with 2-amino-5-phosphonopentanoic acid (AP5) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and using high [Cl − ] intracellular pipette solution. White dotted line represents 0pA level. Application of 1 µmol L -1 baclofen results in a complete silencing of the oscillating activity. Ab-c, Voltage clamp traces at higher temporal resolution reveal a decrease in the frequency of sIPSCs during application of baclofen (Ac) compared to the oscillation UP state under control conditions (Ab). Ad-e, Decreased sIPSC frequency in the presence of baclofen (Ae) is also observed compared to the DOWN state (Ad). Af, Quantification of sIPSC frequency comparing total oscillation (under control conditions) and under baclofen application (grey lines represent individual data points; black line represents the mean ± SEM). Ag, Quantification of sIPSC frequency comparing UP state (control) and under baclofen application. Ah, Quantification of sIPSC frequency comparing DOWN state (control) and under baclofen application. Ai, The amplitude of sIPSCs remains unaffected by application of baclofen. Ba, Voltage clamp recording of a TIDA neurone in vitro, clamped at −80 mV. Recordings of spontaneous excitatory PSCs (sEPSCs) were performed when blocking GABAergic synaptic transmission with gabazine and using KGlu intracellular pipette solution. Bb-c, At greater temporal resolution a decrease is seen in sEPSC frequency during application of baclofen (Bc) compared to the oscillation UP state under control conditions (Bb) in the current traces. Bd-f, Quantification of sEPSC frequency comparing full oscillation (Bd), UP states (Be) and DOWN state (Bf) under control conditions, with baclofen application. (Figure organised as Af.) Bg, The amplitude of sEPSCs remains unaffected by application of baclofen. *P ˂ 0.05, **P ˂ 0.01, and ***P ˂ 0.001, ns, not significant bath application conditions), allowing for a fine-tuning at separate cell compartments.
In the present study, we focused on the peripubertal male TIDA system, where the dopaminergic brake on prolactin release is assumed to be less subject to fluctuation than in the female where prolactin rises transiently during pro-oestrus/oestrus 61 and more persistently during lactation. 62 Whether GABA B actions on TIDA neurones are similar or different in the female rodent (or in older males) remains to be investigated. As oscillations can be observed also in TIDA preparations from female rats, 48 the basic physiology of the circuit is, however, likely to be conserved across sex, at least in some aspects.
In summary, the present results demonstrate that TIDA neurones can be powerfully inhibited by GABA B receptors, probably via GIRK-mediated hyperpolarisation. These data suggest that TIDAderived GABA may play a role not exclusive to the receiving end of the parvocellular neuroendocrine neurones (i.e. the pituitary) 19,20 and could also mediate regulation of neuronal network activity at the master level of the endocrine system, the hypothalamic neurosecretory cells. Thus, similar to the cerebral cortex, 63 Figure 5A,B).

ACK N OWLED G EM ENTS
The authors express their gratitude to Dr Paul Williams for expert assistance in designing Figure 5. We also thank the other mem-

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
RA and CB designed the experiments and wrote the manuscript. RA performed and analysed all experiments.

DATA AVA I L A B I L I T Y
The data that support the findings of this study are available from the corresponding author upon reasonable request.