Stefano Zucca

Changes in Expression and Function of Extrasynaptic GABAA Receptors in the Rat Hippocampus during Pregnancy and after Delivery

Pregnancy is associated with changes in mood and anxiety level as well as with marked hormonal fluctuations. Increases in the brain concentrations of neuroactive steroids during pregnancy in rats are accompanied by changes in expression of subunits of the GABA type A receptor (GABAA-R) in the brain. Granule cells of the dentate gyrus (DGGCs) exhibit two components of inhibitory GABAergic transmission: a phasic component mediated by synaptic GABAA-Rs, and a tonic component mediated by extrasynaptic GABAA-Rs. Recordings of GABAergic currents were obtained from hippocampal slices prepared from rats in estrus, at pregnancy day 15 (P15) or P19, or at 2 d after delivery. Exogenous GABA or 3α,5α-THP induced an increase in tonic current in DGGCs that was significantly greater at P19 than in estrus. Neither tonic nor phasic currents were affected by pregnancy in CA1 pyramidal cells. Immunohistochemical analysis revealed a marked increase in the abundance of the δ subunit of the GABAA-R and a concomitant decrease in that of the γ2 subunit in the hippocampus at P19. Expression of the α4 subunit did not change during pregnancy but was increased 2 d after delivery. Treatment of rats from P12 to P18 with the 5α-reductase inhibitor finasteride prevented the changes in tonic current and in δ and γ2 subunit expression normally apparent at P19. These data suggest that the number of extrasynaptic GABAA-Rs is increased in DGGCs during late pregnancy as a consequence of the associated marked fluctuations in the brain levels of neuroactive steroids.

Alcohol Exposure Decreases CREB Binding Protein Expression and Histone Acetylation in the Developing Cerebellum

Background Fetal alcohol exposure affects 1 in 100 children making it the leading cause of mental retardation in the US. It has long been known that alcohol affects cerebellum development and function. However, the underlying molecular mechanism is unclear. Methodology/Principal Findings We demonstrate that CREB binding protein (CBP) is widely expressed in granule and Purkinje neurons of the developing cerebellar cortex of naïve rats. We also show that exposure to ethanol during the 3rd trimester-equivalent of human pregnancy reduces CBP levels. CBP is a histone acetyltransferase, a component of the epigenetic mechanism controlling neuronal gene expression. We further demonstrate that the acetylation of both histone H3 and H4 is reduced in the cerebellum of ethanol- treated rats. Conclusions/Significance These findings indicate that ethanol exposure decreases the expression and function of CBP in the developing cerebellum. This effect of ethanol may be responsible for the motor coordination deficits that characterize fetal alcohol spectrum disorders.

Low Concentrations of Alcohol Inhibit BDNF-Dependent GABAergic Plasticity via L-type Ca2+ Channel Inhibition in Developing CA3 Hippocampal Pyramidal Neurons

Fetal alcohol spectrum disorder (FASD) is associated with learning and memory alterations that could be, in part, a consequence of hippocampal damage. The CA3 hippocampal subfield is one of the regions affected by ethanol (EtOH), including exposure during the third trimester-equivalent (i.e., neonatal period in rats). However, the mechanism of action of EtOH is poorly understood. In CA3 pyramidal neurons from neonatal rats, dendritic BDNF release causes long-term potentiation of the frequency of GABAA receptor-mediated spontaneous postsynaptic currents (LTP-GABAA) and this mechanism is thought to play a role in GABAergic synapse maturation. Here, we show that short- and long-term exposure of neonatal male rats to low EtOH concentrations abolishes LTP-GABAA by inhibiting L-type voltage-gated Ca2+ channels. These findings support the recommendation that even light drinking should be avoided during pregnancy.

Ethanol Decreases Purkinje Neuron Excitability by Increasing GABA Release in Rat Cerebellar Slices

Cerebellar Purkinje neurons (PNs) receive inhibitory GABAergic input from stellate and basket cells, which are located in the outer and inner portions of the molecular layer, respectively. Ethanol (EtOH) was recently shown to increase GABAergic transmission at PNs via a mechanism that involves enhanced calcium release from presynaptic internal stores (J Pharmacol Exp Ther 323:356–364, 2007). Here, we further characterized the effect of EtOH on GABA release and assessed its impact on PN excitability. Using whole-cell patch-clamp electrophysiological techniques in cerebellar vermis parasagittal slices, we found that EtOH acutely increases the frequency but not the amplitude or half-width of miniature and spontaneous inhibitory postsynaptic currents (IPSCs). EtOH significantly increased the amplitude and decreased the paired pulse ratio of IPSCs evoked by stimulation in the outer but not inner molecular layer. In current clamp, EtOH decreased both the amplitude of excitatory postsynaptic potentials evoked in PNs by granule cell axon stimulation and the number of action potentials triggered by these events; these effects depended on GABAA receptor activation because they were not observed in presence of bicuculline. Loose-patch cell-attached PN recordings revealed that neither the spontaneous action potential firing frequency nor the coefficient of variation of the interspike interval was altered by acute EtOH exposure. These findings suggest that EtOH differentially affects GABAergic transmission at stellate cell- and basket cell-to-PN synapses and that it modulates PN firing triggered by granule cell axonal input. These effects could be in part responsible for the cerebellar impairments associated with acute EtOH intoxication.

Role of Striatal Cholinergic Interneurons in Set-Shifting in the Rat

The ability to change strategies in different contexts is a form of behavioral flexibility that is crucial for adaptive behavior. The striatum has been shown to contribute to certain forms of behavioral flexibility such as reversal learning. Here we report on the contribution of striatal cholinergic interneurons—a key element in the striatal neuronal circuit—to strategy set-shifting in which an attentional shift from one stimulus dimension to another is required. We made lesions of rat cholinergic interneurons in dorsomedial or ventral striatum using a specific immunotoxin and investigated the effects on set-shifting paradigms and on reversal learning. In shifting to a set that required attention to a previously irrelevant cue, lesions of dorsomedial striatum significantly increased the number of perseverative errors. In this condition, the number of never-reinforced errors was significantly decreased in both types of lesions. When shifting to a set that required attention to a novel cue, rats with ventral striatum lesions made more perseverative errors. Neither lesion impaired learning of the initial response strategy nor a subsequent switch to a new strategy when response choice was indicated by a previously relevant cue. Reversal learning was not affected. These results suggest that in set-shifting the striatal cholinergic interneurons play a fundamental role, which is dissociable between dorsomedial and ventral striatum depending on behavioral context. We propose a common mechanism in which cholinergic interneurons inhibit neurons representing the old strategy and enhance plasticity underlying exploration of a new rule.

Pauses in cholinergic interneuron firing exert an inhibitory control on striatal output in vivo

The cholinergic interneurons (CINs) of the striatum are crucial for normal motor and behavioral functions of the basal ganglia. Striatal CINs exhibit tonic firing punctuated by distinct pauses. Pauses occur in response to motivationally significant events, but their function is unknown. Here we investigated the effects of pauses in CIN firing on spiny projection neurons (SPNs) – the output neurons of the striatum – using in vivo whole cell and juxtacellular recordings in mice. We found that optogenetically-induced pauses in CIN firing inhibited subthreshold membrane potential activity and decreased firing of SPNs. During pauses, SPN membrane potential fluctuations became more hyperpolarized and UP state durations became shorter. In addition, short-term plasticity of corticostriatal inputs was decreased during pauses. Our results indicate that, in vivo, the net effect of the pause in CIN firing on SPNs activity is inhibition and provide a novel mechanism for cholinergic control of striatal output.

Cholinergic interneurons in the rat striatum modulate substitution of habits

Behavioural flexibility is crucial for adaptive behaviour, and recent evidence suggests that cholinergic interneurons of the striatum play a distinct role. Previous studies of cholinergic function have focused on strategy switching by the dorsomedial or ventral striatum. We here investigated whether cholinergic interneurons in the dorsolateral striatum play a similar role at the level of switching of habitual responses. Because the dorsolateral striatum is particularly involved in habitual responding, we developed a habit substitution task that involved switching habitual lever‐press responses to one side to another. We first measured the effect of cholinergic activation in the dorsolateral striatum on this task. Chemogenetic activation of cholinergic interneurons caused an increase in the response rate for the substituted response that was significantly greater than the increase normally seen in control animals. The increase was due to burst‐like responses with shorter inter‐press intervals. However, there was no effect on inhibiting the old habit, or on habitual responding that did not require a switch. There was also no effect on lever‐press performance and its reversal before lever‐press responses became habitual. Conversely, neurochemically specific ablation of cholinergic interneurons did not significantly change habitual responding or response substitution. Thus, activation –but not ablation –of cholinergic interneurons in the dorsolateral striatum modulates expression of a new habit when an old habit is replaced by a new one. Together with previous work, this suggests that striatal cholinergic interneurons facilitate behavioural flexibility in both dorsolateral striatum in addition to dorsomedial and ventral striatum.

New Variations for Strategy Set-shifting in the Rat

Behavioral flexibility is crucial for survival in changing environments. Broadly defined, behavioral flexibility requires a shift of behavioral strategy based on a change in governing rules. We describe a strategy set-shifting task that requires an attentional shift from one stimulus dimension to another. The paradigm is often used for testing cognitive flexibility in primates. However, the rodent version has not been as extensively developed. We have recently extended an established set-shifting task in the rat1 by requiring attention to different stimuli according to context. All the experimental conditions required animals to choose either a left or right lever. Initially, all animals had to choose on the basis of the location of the lever. Subsequently, a change in the rule occurred, which required a shift in set from location-based rule to a rule in which the correct lever was indicated by a light cue. We compared performance on three different versions of the task, in which the light stimulus was either novel, previously relevant, or previously irrelevant. We found that specific neurochemical lesions selectively impaired the ability to make particular types of set shift as measured by the performance on the different versions of the task.

Cholinergic mechanisms in adaptive behaviour

From April 14th to 15th, 2016, a group of researchers investigating the role of acetylcholine within brain circuits responsible for learning and behaviour gathered in Okinawa, Japan, for a mini-symposium hosted by the Okinawa Institute of Science and Technology Graduate University. The aim was to work towards an integrated concept of the contribution of acetylcholine to higher brain functions, and the underlying neural mechanisms, by integrating the most recent results into a coherent concept of the role of acetylcholine in overall brain function, focusing on the basal ganglia in interaction with the thalamus and cerebral cortex. This Special Section of the European Journal of Neuroscience brings together several papers based on invited talks presented at this mini-symposium. Acetylcholine has long been known to play a crucial role in adaptive behaviour, but the limited access to the cholinergic neurons which release acetylcholine has limited progress. The recent availability of genetic approaches to targeting cholinergic neurons for experimental study, combined with sophisticated electrophysiological, behavioural and imaging approaches, has led to an explosion of new findings. The field is on the cusp of major advances in understanding the role of acetylcholine at cellular, circuit and system levels. In this Special Section, Tanimura et al. give a perspective from the Surmeier laboratory on the part cholinergic interneurons play in normal striatal physiology, and how their role changes in Parkinson’s disease. Findings from new methodologies such as optogenetics and monosynaptic rabies virus mapping are discussed. Kobayashi et al. report on the effects that immunotoxin lesions of cholinergic interneurons in the dorsomedial striatum have on behavioural flexibility. Aoki et al. write about the role of cholinergic interneurons in habit substitution, investigated by chemogenetic activation of cholinergic interneurons in the dorsolateral striatum. Heath and colleagues from the Balleine laboratory report on the neurochemical drivers underlying the accumulation of delta-opioid receptors at the somatic membrane of cholinergic interneurons in the nucleus accumbens shell, which is required for predictive learning to influence the subsequent choice between goal-directed actions. Bridging between rodent neurochemistry and human brain function, Bell et al., from the Christakou laboratory report pioneering work using proton magnetic resonance spectroscopy as a noninvasive procedure to estimate acetylcholine levels from the levels of choline in humans performing a reversal learning task. From this encouraging start, we hope will grow a convergence of concepts, methods and experimental studies addressing the enigmatic role of the cholinergic interneurons in the striatum. For many decades, progress has been limited by our lack of tools to manipulate and measure their activity. The availability of powerful new technologies provides an opportunity to extend our understanding of this remarkable neural system.