GABAB receptor antagonism from birth to weaning permanently modifies Kiss1 expression in hypothalamus and gonads in mice

Introduction: The kisspeptin gene Kiss1 is expressed in two hypothalamic areas: anteroventral periventricular nucleus/periventricular nucleus (AVPV/PeN) and arcuate nucleus (ARC), and also in gonads. Evidences suggest that gamma-amino butyric acid B receptors (GABAB) signaling can regulate Kiss1 expression. Here we inhibited GABAB signaling from PND2-PND21 and evaluated the hypothalamic-pituitary-gonadal (HPG) axis. Methods: BALB/c mice were treated on postnatal days 2-21 (PND2-PND21) with CGP55845 (GABAB antagonist), and evaluated in PND21 and adulthood: gene expression (qPCR) in hypothalamus and gonads, hormones by radioimmunoassay, gonad histochemistry (H&E), puberty onset, estrous cycles. Results: At PND21, CGP inhibited Kiss1 and Tac2 and increased Pdyn and Gabbr1 in the ARC of both sexes and decreased Th only in female AVPV/PeN. Serum follicle-stimulating hormone (FSH) and testis weight decreased in CGP-males and puberty onset was delayed. In adults, Kiss1, Tac2, Pdyn, Pgr, Cyp19a1, Gad1 were downregulated, while Gabbr1 was upregulated in the ARC of both sexes. In the AVPV/PeN, Kiss1, Th, Cyp19a1 and Pgr decreased while Gad1 increased in CGP-females, whereas Cyp19a1 increased in CGP-males. Serum FSH increased in CGP-males while prolactin increased in CGP-females. Testosterone and progesterone increased in ovaries from CGP-females, in which Kiss1, Cyp19a1 and Esr1 were downregulated while Hsd3b2 was upregulated, together with increased atretic and decreased ovulatory follicles. Testes from CGP-males showed decreased progesterone, increased Gabbr1, Kiss1, Kiss1r, Esr2 and decreased Cyp19a1 and clear signs of seminiferous tubules atrophy. Conclusion: These results demonstrate that appropriate GABAB signaling during this critical prepubertal period is necessary for the normal development of the HPG axis.

Effectiveness and relationship between biased and unbiased measures of dopamine release and clearance

Fast-scan cyclic voltammetry (FSCV) is an effective tool for measuring dopamine (DA) release and clearance throughout the brain, including the ventral and dorsal striatum. Striatal DA terminals are abundant with signals heavily regulated by release machinery and the dopamine transporter (DAT). Peak height is a common method for measuring release but can be affected by changes in clearance. The Michaelis-Menten model has been a standard in measuring DA clearance, but requires experimenter fitted modeling subject to experimenter bias. The current study presents the use of the first derivative (velocity) of evoked DA signals as an alternative approach for measuring dopamine release and clearance and can be used to distinguish the two measures. Maximal upwards velocity predicts reductions in DA peak height due to D2 and GABAB receptor stimulation and by alterations in calcium concentrations. The Michaelis-Menten maximal velocity (Vmax) measure, an approximation for DAT numbers, predicted maximal downward velocity in slices and in vivo. Dopamine peak height and upward velocity were similar between wildtype C57 (WT) and DAT knock out (DATKO) mice. In contrast, downward velocity was considerably reduced and exponential decay (tau) was increased in DATKO mice, supporting use of both measures for changes in DAT activity. In slices, the competitive DAT inhibitors cocaine, PTT and WF23 increased peak height and upward velocity differentially across increasing concentrations, with PTT and cocaine reducing these measures at high concentrations. Downward velocity and tau values decreased and increased respectively across concentrations, with greater potency and efficacy observed with WF23 and PTT. In vivo recordings demonstrated similar effects of WF23 and PTT on measures of release and clearance. Tau was a more sensitive measure at low concentrations, supporting its use as a surrogate for the Michaelis-Menten measure of apparent affinity (Km). Together, these results inform on the use of these measures for DA release and clearance.

GABABR Modulation of Electrical Synapses and Plasticity in the Thalamic Reticular Nucleus

Two distinct types of neuronal activity result in long-term depression (LTD) of electrical synapses, with overlapping biochemical intracellular signaling pathways that link activity to synaptic strength, in electrically coupled neurons of the thalamic reticular nucleus (TRN). Because components of both signaling pathways can also be modulated by GABAB receptor activity, here we examined the impact of GABAB receptor activation on the two established inductors of LTD in electrical synapses. Recording from patched pairs of coupled rat neurons in vitro, we show that GABAB receptor inactivation itself induces a modest depression of electrical synapses and occludes LTD induction by either paired bursting or metabotropic glutamate receptor (mGluR) activation. GABAB activation also occludes LTD from either paired bursting or mGluR activation. Together, these results indicate that afferent sources of GABA, such as those from the forebrain or substantia nigra to the reticular nucleus, gate the induction of LTD from either neuronal activity or afferent glutamatergic receptor activation. These results add to a growing body of evidence that the regulation of thalamocortical transmission and sensory attention by TRN is modulated and controlled by other brain regions. Significance: We show that electrical synapse plasticity is gated by GABAB receptors in the thalamic reticular nucleus. This effect is a novel way for afferent GABAergic input from the basal ganglia to modulate thalamocortical relay and is a possible mediator of intra-TRN inhibitory effects.

The Novel Positive Allosteric Modulator of the GABAB Receptor, KK-92A, Suppresses Alcohol Self-Administration and Cue-Induced Reinstatement of Alcohol Seeking in Rats

Positive allosteric modulators (PAMs) of the GABAB receptor (GABAB PAMs) are of interest in the addiction field due to their ability to suppress several behaviors motivated by drugs of abuse. KK-92A is a novel GABAB PAM found to attenuate intravenous self-administration of nicotine and reinstatement of nicotine seeking in rats. This present study was aimed at extending to alcohol the anti-addictive properties of KK-92A. To this end, Sardinian alcohol-preferring rats were trained to lever-respond for oral alcohol (15% v/v) or sucrose (0.7% w/v) under the fixed ratio (FR) 5 (FR5) schedule of reinforcement. Once lever-responding behavior had stabilized, rats were exposed to tests with acutely administered KK-92A under FR5 and progressive ratio schedules of reinforcement and cue-induced reinstatement of previously extinguished alcohol seeking. KK-92A effect on spontaneous locomotor activity was also evaluated. Treatment with 10 and 20 mg/kg KK-92A suppressed lever-responding for alcohol, amount of self-administered alcohol, and breakpoint for alcohol. Treatment with 20 mg/kg KK-92A reduced sucrose self-administration. Combination of per se ineffective doses of KK-92A (2.5 mg/kg) and the GABAB receptor agonist, baclofen (1 mg/kg), reduced alcohol self-administration. Treatment with 5, 10, and 20 mg/kg KK-92A suppressed reinstatement of alcohol seeking. Only treatment with 80 mg/kg KK-92A affected spontaneous locomotor activity. These results demonstrate the ability of KK-92A to inhibit alcohol-motivated behaviors in rodents and confirm that these effects are common to the entire class of GABAB PAMs. The remarkable efficacy of KK-92A is discussed in terms of its ago-allosteric properties.