The functional implications of transient receptor potential melastatin 3 (TRPM3) activation, the most recently described member of the melastatin subfamily of cation permeable TRP channels, have begun to be elucidated in recent years. The discovery of TRPM3 activation by the steroid pregnenolone sulfate (PregS) has shed new light on the physiological role of this channel. For example, TRPM3 activation enhances insulin secretion from β pancreatic cells, induces contraction of vascular smooth muscle, and is also involved in the detection of noxious heat. Although TRPM3 expression has been detected in several regions of the developing and mature brain, little is known about the roles of TRPM3 in brain physiology. Here, we demonstrate the abundant expression of TRPM3 steroid-sensitive channels in the developing cerebellar cortex. We also show that TRPM3-like channels are expressed at glutamatergic synapses in neonatal Purkinje cells (PCs). We recently showed that PregS potentiates spontaneous glutamate release onto neonatal PCs during a period of active glutamatergic synapse formation; we now show that this effect of PregS is mediated by TRPM3-like channels. Mefenamic acid, a recently discovered TRPM3 antagonist, blocked the effect of PregS on glutamate release. The PregS effect on glutamate release was mimicked by other TRPM3 agonists (nifedipine and epipregnanolone sulfate) but not by a TRMP3-inactive steroid (progesterone). Our findings identify TRPM3 channels as novel modulators of glutamatergic transmission in the developing brain.

Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ~60 mM (~0.28 g/dl) during the 4 hours of exposure. EtOH levels gradually decreased to baseline 8 hrs after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased the expression of δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABAA receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats.

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Development of cerebellar Purkinje cells (PCs) is modulated by neuroactive steroids. Developing hippocampal pyramidal neurons retrogradely release a pregnenolone sulfate (PregS)-like neurosteroid that may contribute to glutamatergic synapse stabilization. We hypothesized that PregS could exert a similar effect on developing PCs. To test this hypothesis, we performed whole-cell patch-clamp recordings from PCs in acute cerebellar vermis slices from neonatal rats. PregS induced a robust (~3,000 %) and reversible increase in AMPA receptor-mediated miniature excitatory postsynaptic current (AMPA-mEPSC) frequency without affecting the amplitude, timeto-rise, or half-width of these events. PregS also increased the frequency of GABA A receptormediated miniature postsynaptic currents but to a significantly lesser extent (<100%). The PregSinduced increase of AMPA-mEPSC frequency was not significantly decreased by antagonists of receptors (NMDA, glycine, α7 nicotinic acetylcholine, and σ1) that have been shown to modulate glutamatergic transmission at PCs and/or mediate the actions of PregS on neurotransmitter release. Ca 2+ chelation experiments suggested that PregS acts by increasing presynaptic terminal [Ca 2+ ] i , an effect that is independent of voltage-gated Ca 2+ channels, but is blocked by the antagonist of transient receptor potential (TRP) channels, La 3+ . PregS also increased the amplitude of EPSCs evoked by climbing fiber (CF) stimulation and decreased the paired-pulse ratio of these events. Neither CF-nor parallel fiber-evoked EPSCs were affected by PregS in slices from juvenile rats. These results suggest that glutamate release at CF-to-PC synapses is an important target of PregS in the neonatal cerebellar cortex, an effect that may play a role in the refinement of these synapses.

Long-term consumption of ethanol affects cortical areas that are important for learning and memory, cognition, and decision-making. Deficits in cortical function may contribute to alcohol-abuse disorders by impeding an individual’s ability to control drinking. Previous studies from this laboratory show that acute ethanol reduces activity of lateral orbitofrontal cortex (LOFC) neurons while chronic exposure impairs LOFC-dependent reversal learning and induces changes in LOFC excitability. Despite these findings, the role of LOFC neurons in ethanol consumption is unknown. To address this issue, we examined ethanol drinking in adult C57Bl/6J mice that received an excitotoxic lesion or viral injection of the inhibitory DREADD (designer receptor exclusively activated by designer drug) into the LOFC. No differences in ethanol consumption were observed between sham and lesioned mice during access to increasing concentrations of ethanol (3–40%) every other day for 7 weeks. Adulterating the ethanol solution with saccharin (0.2%) or quinine (0.06 mM) enhanced or inhibited, respectively, consumption of the 40% ethanol solution similarly in both groups. Using a chronic intermittent ethanol (CIE) vapor exposure model that produces dependence, we found no difference in baseline drinking between sham and lesioned mice prior to vapor treatments. CIE enhanced drinking in both groups as compared to air-treated animals and CIE treated lesioned mice showed an additional increase in ethanol drinking as compared to CIE sham controls. This effect persisted during the first week when quinine was added to the ethanol solution but consumption decreased to control levels in CIE lesioned mice in the following 2 weeks. In viral injected mice, baseline drinking was not altered by expression of the inhibitory DREADD receptor and repeated cycles of CIE exposure enhanced drinking in DREADD and virus control groups. Consistent with the lesion study, treatment with clozapine-N-oxide (CNO) further enhanced consumption only in CIE exposed DREADD mice with no change in air-treated mice. These results suggest that the LOFC is not critical for the initiation and maintenance of ethanol drinking in non-dependent mice, but may regulate the escalated drinking observed during dependence.