Consensus Paper: Cerebellum and Emotion.

Over the past three decades, insights into the role of the cerebellum in emotional processing have substantially increased. Indeed, methodological refinements in cerebellar lesion studies and major technological advancements in the field of neuroscience are in particular responsible to an exponential growth of knowledge on the topic. It is timely to review the available data and to critically evaluate the current status of the role of the cerebellum in emotion and related domains. The main aim of this article is to present an overview of current facts and ongoing debates relating to clinical, neuroimaging, and neurophysiological findings on the role of the cerebellum in key aspects of emotion. Experts in the field of cerebellar research discuss the range of cerebellar contributions to emotion in nine topics. Topics include the role of the cerebellum in perception and recognition, forwarding and encoding of emotional information, and the experience and regulation of emotional states in relation to motor, cognitive, and social behaviors. In addition, perspectives including cerebellar involvement in emotional learning, pain, emotional aspects of speech, and neuropsychiatric aspects of the cerebellum in mood disorders are briefly discussed. Results of this consensus paper illustrate how theory and empirical research have converged to produce a composite picture of brain topography, physiology, and function that establishes the role of the cerebellum in many aspects of emotional processing.

Genetic deletion of fibroblast growth factor 14 recapitulates phenotypic alterations underlying cognitive impairment associated with schizophrenia.

Cognitive processing is highly dependent on the functional integrity of gamma-amino-butyric acid (GABA) interneurons in the brain. These cells regulate excitability and synaptic plasticity of principal neurons balancing the excitatory/inhibitory tone of cortical networks. Reduced function of parvalbumin (PV) interneurons and disruption of GABAergic synapses in the cortical circuitry result in desynchronized network activity associated with cognitive impairment across many psychiatric disorders, including schizophrenia. However, the mechanisms underlying these complex phenotypes are still poorly understood. Here we show that in animal models, genetic deletion of fibroblast growth factor 14 (Fgf14), a regulator of neuronal excitability and synaptic transmission, leads to loss of PV interneurons in the CA1 hippocampal region, a critical area for cognitive function. Strikingly, this cellular phenotype associates with decreased expression of glutamic acid decarboxylase 67 (GAD67) and vesicular GABA transporter (VGAT) and also coincides with disrupted CA1 inhibitory circuitry, reduced in vivo gamma frequency oscillations and impaired working memory. Bioinformatics analysis of schizophrenia transcriptomics revealed functional co-clustering of FGF14 and genes enriched within the GABAergic pathway along with correlatively decreased expression of FGF14, PVALB, GAD67 and VGAT in the disease context. These results indicate that Fgf14(-/-) mice recapitulate salient molecular, cellular, functional and behavioral features associated with human cognitive impairment, and FGF14 loss of function might be associated with the biology of complex brain disorders such as schizophrenia.

Auditory cortex involvement in emotional learning and memory.

Emotional memories represent the core of human and animal life and drive future choices and behaviors. Early research involving brain lesion studies in animals lead to the idea that the auditory cortex participates in emotional learning by processing the sensory features of auditory stimuli paired with emotional consequences and by transmitting this information to the amygdala. Nevertheless, electrophysiological and imaging studies revealed that, following emotional experiences, the auditory cortex undergoes learning-induced changes that are highly specific, associative and long lasting. These studies suggested that the role played by the auditory cortex goes beyond stimulus elaboration and transmission. Here, we discuss three major perspectives created by these data. In particular, we analyze the possible roles of the auditory cortex in emotional learning, we examine the recruitment of the auditory cortex during early and late memory trace encoding, and finally we consider the functional interplay between the auditory cortex and subcortical nuclei, such as the amygdala, that process affective information. We conclude that, starting from the early phase of memory encoding, the auditory cortex has a more prominent role in emotional learning, through its connections with subcortical nuclei, than is typically acknowledged.

Evidence for a common progenitor of epithelial and mesenchymal components of the liver.

Tissues of the adult organism maintain the homeostasis and respond to injury by means of progenitor/stem cell compartments capable to give rise to appropriate progeny. In organs composed by histotypes of different embryological origins (e.g. the liver), the tissue turnover may in theory involve different stem/precursor cells able to respond coordinately to physiological or pathological stimuli. In the liver, a progenitor cell compartment, giving rise to hepatocytes and cholangiocytes, can be activated by chronic injury inhibiting hepatocyte proliferation. The precursor compartment guaranteeing turnover of hepatic stellate cells (HSCs) (perisinusoidal cells implicated with the origin of the liver fibrosis) in adult organ is yet unveiled. We show here that epithelial and mesenchymal liver cells (hepatocytes and HSCs) may arise from a common progenitor. Sca+ murine progenitor cells were found to coexpress markers of epithelial and mesenchymal lineages and to give rise, within few generations, to cells that segregate the lineage-specific markers into two distinct subpopulations. Notably, these progenitor cells, clonally derived, when transplanted in healthy livers, were found to generate epithelial and mesenchymal liver-specific derivatives (i.e. hepatocytes and HSCs) properly integrated in the liver architecture. These evidences suggest the existence of a 'bona fide' organ-specific meso-endodermal precursor cell, thus profoundly modifying current models of adult progenitor commitment believed, so far, to be lineage-restricted. Heterotopic transplantations, which confirm the dual differentiation potentiality of those cells, indicates as tissue local cues are necessary to drive a full hepatic differentiation. These data provide first evidences for an adult stem/precursor cell capable to differentiate in both parenchymal and non-parenchymal organ-specific components and candidate the liver as the instructive site for the reservoir compartment of HSC precursors as yet non-localized in the adult.

Involvement of cerebellum in emotional behavior.

In the last decade a growing body of data revealed that the cerebellum is involved in the regulation of the affective reactions as well as in forming the association between sensory stimuli and their emotional values. In humans, cerebellar areas around the vermis are activated during mental recall of emotional personal episodes and during learning of a CS-US association. Lesions of the cerebellar vermis may affect retention of a fear memory without altering baseline motor/autonomic responses to the frightening stimuli in both human and animal models. Reversible inactivation of the vermis during the consolidation period impairs retention of fear memory in rodents. Recent findings demonstrate that long-term potentiation (LTP) of synapses in the cerebellar cortex occurs in relation to associative fear learning similar to previously reported data in the hippocampus and amygdala. Plastic changes affect both excitatory and inhibitory synapses. This concomitant potentiation allows the cerebellar cortical network to detect coincident inputs, presumably conveying sensorial stimuli, with better efficacy by keeping the time resolution of the system unchanged. Collectively, these data suggest that the vermis participates in forming new CS-US association and translate an emotional state elaborated elsewhere into autonomic and motor responses.

Cerebellum and emotional behavior.

Fear conditioning involves learning that a previously neutral stimulus (CS) predicts an aversive unconditioned stimulus (US). Lesions of the cerebellar vermis may affect fear memory without altering baseline motor/autonomic responses to the frightening stimuli. Reversible inactivation of the vermis during the consolidation period impairs retention of fear memory. In patients with medial cerebellar lesions conditioned bradycardia is impaired. In humans, cerebellar areas around the vermis are activated during mental recall of emotional personal episodes, if a loved partner receives a pain stimulus, and during learning of a CS-US association. Moreover, patients with cerebellar stroke may fail to show overt emotional changes. In such patients, however, the activity of several areas, including ventromedial prefrontal cortex, anterior cingulate, pulvinar and insular cortex, is significantly increased relative to healthy subjects when exposed to frightening stimuli. Therefore, other structures may serve to maintain fear response after cerebellar damage. These data indicate that the vermis is involved in the formation of fear memory traces. We suggest that the vermis is not only involved in regulating the autonomic/motor responses, but that it also participates in forming new CS-US associations thus eliciting appropriate responses to new stimuli or situations. In other words, the cerebellum may translate an emotional state elaborated elsewhere into autonomic and motor responses.

Lentiviral transduction of human postnatal skeletal (stromal, mesenchymal) stem cells: in vivo transplantation and gene silencing.

Systems for gene transfer and silencing in human skeletal stem cells (hSSCs, also stromal or mesenchymal stem cells) are important for addressing critical issues in basic hSSC and skeletal biology and for developing gene therapy strategies for treatment of skeletal diseases. Whereas recent studies have shown the efficacy of lentiviral transduction for gene transfer in hSSCs in vitro, no study has yet proven that lentivector-transduced hSSCs retain their distinctive organogenic potential in vivo, as probed by in vivo transplantation assays. Therefore, in addition to analyzing the in vitro growth and differentiation properties of hSSCs transduced with advanced-generation lentivectors, we ectopically transplanted LV-eGFP-transduced hSSCs (along with an osteoconductive carrier) in the subcutaneous tissue of immunocompromised mice. eGFP-transduced cells formed heterotopic ossicles, generating osteoblasts, osteocytes, and stromal cells in vivo, which still expressed GFP at 2 months after transplantation. eGFP-expressing cells could be recovered from the ossicles 8 weeks posttransplantation and reestablished in culture as viable and proliferating cells. Further, we investigated the possibility of silencing individual genes in hSSCs using lentivectors encoding short hairpin precursors of RNA interfering sequences under the control of the Pol-III-dependent H1 promoter. Significant long-term silencing of both lamin A/C and GFP (an endogenous gene and a transgene, respectively) was obtained with lentivectors encoding shRNAs. These data provide the basis for analysis of the effect of gene knockdown during the organogenesis of bone in the in vivo transplantation system and for further studies on the silencing of alleles carrying dominant, disease-causing mutations.

Membrane excitability and fear conditioning in cerebellar Purkinje cell.

In a previous study it has been demonstrated that fear conditioning is associated with a long-lasting potentiation of parallel fiber to Purkinje cell synaptic transmission in vermal lobules V and VI. Since modifications of intrinsic membrane properties have been suggested to mediate some forms of memory processes, we investigated possible changes of Purkinje cell intrinsic properties following the same learning paradigm and in the same cerebellar region. By means of the patch clamp technique, Purkinje cell passive and active membrane properties were evaluated in slices prepared from rats 10 min or 24 h after fear conditioning and in slices from control naïve animals. None of the evaluated parameters (input resistance, inward rectification, maximal firing frequency and the first inter-spike interval, post-burst afterhyperpolarization, action potential threshold and amplitude, action potential afterhyperpolarization) was significantly different between the three studied groups also in those cells where parallel fiber-Purkinje cell synapse was potentiated. Our results show that fear learning does not affect the intrinsic membrane properties involved in Purkinje cell firing. Therefore, at the level of Purkinje cell the plastic change associated with fear conditioning is specifically restricted to synaptic efficacy.

The presence of astrocytes enhances beta amyloid-induced neurotoxicity in hippocampal cell cultures.

A characteristic feature of neuritic plaques in Alzheimer's disease is represented by the presence of activated astrocytes, surrounding dystrophic neurons and beta-amyloid deposition. To explore the role of astrocytes in in vitro beta-amyloid neurotoxicity, we studied the effect of beta-amyloid treatment in hippocampal neurons in two different cell models: pure cultures, where neurons were grown in absence of astrocytes and mixed cultures, where neurons were seeded on a confluent layer of astrocytes. We evaluated two characteristic aspects of in vitro beta-amyloid neurotoxicity: reduction of cell viability and degeneration of the neuritic tree. We demonstrated that neurons growing on astrocytes were more prone to the detrimental effect of the amyloid peptide, with respect to neurons grown in absence of the glial component. Our results support the hypothesis that beta-amyloid-astrocyte interaction can adversely condition neurons and contribute to neuronal damage in Alzheimer's disease.

Pituitary adenylate cyclase-activating polypeptide hormone (PACAP) at very low dosages improves memory in the rat.

To ascertain whether very low dosages of pituitary adenylate cyclase-activating polypeptide (PACAP) influence learning in mammals, immediately after the acquisition trial of a passive avoidance response (PAR) paradigm, PACAP-38 was administered intracerebroventricularly at increasing dosages (0, 0.02, 0.2, 2, 20, and 200 ng in 10 microl saline) to different groups of rats. The mnemonic effects were measured by means of retention testing 48 and 96 h later. At intermediate PACAP-38 concentrations there was a significant mnemonic improvement of the PAR. The maximal effect was observed after the 0.2-ng PACAP-38 administration (longer step-through latencies). There was a lesser effect at the subsequent higher concentration, 2 ng. Higher dosages had no effects. It is concluded that PACAP-38 acts as an enhancer of mammalian mnemonic processes even at very low dosages. The positive effect follows an inverted U-shaped dose-response curve. The results may be of interest for the therapy of some neuropathological conditions.

Long-lasting hippocampal potentiation and contextual memory consolidation.

In order to ascertain whether there are hippocampal electrophysiological modifications specifically related to memory, exploratory activity and emotional stress, extracellular electrical activity was recorded in hippocampal slices prepared from the brains of male adult rats. Several groups of animals were employed: (i) rats which had freely explored the experimental apparatus (8 min exposure); (ii) rats which had been subjected, in the same apparatus, to a fear conditioning paradigm training entailing the administration of aversive electrical footshocks (8 min exposure); (iii) rats to which the same number of aversive shocks had been administered in the same apparatus, but temporally compressed so as to make difficult the association between painful stimuli and the apparatus (30 s exposure); (iv) naïve rats never placed in the apparatus. Half of the rats from each treatment group were used for retrieval testing and the other half for hippocampal excitability testing. The conditioned freezing response was exhibited for no less than 4 weeks. Hippocampal excitability was measured by means of input-output curves (IOC) and paired-pulse facilitation curves (PPF). Retrieval testing or brain slices preparation were performed at increasing delays after the training sessions: immediately afterwards or after 1, 7 or 28 days. Only the rats subjected to the fear conditioning training exhibited freezing when placed again in the apparatus (retrieval testing). It was found that IOCs, with respect to naïve rats, increased in the conditioned animals up to the 7-day delay. In free exploration animals the IOCs increased only immediately after the training session. In all other rats no modification of the curves was observed. IOC increases do not appear to imply presynaptic transmitter release modifications, because they were not accompanied by PPF modifications. In conclusion, a clear-cut correlation was found between the increase in excitability of the Schaffer collateral-CA1 dendrite synapses and freezing response consolidation.

CAMKII inhibition in the parabrachial nuclei elicits conditioned taste aversion in rats.

The conditioned taste aversion (CTA) paradigm was used to assess the role of Ca(2+)/calmodulin-dependent protein kinase (CAMKII) in associative learning. KN62, a specific inhibitor of CAMKII, was injected into the parabrachial nuclei (PBN) either immediately after saccharin drinking (CS) or after saccharin drinking and i.p. injection of LiCl (US). Injection of KN62 into the PBN after saccharin drinking elicited clear CTA (Exp. 1). This effect was dosage-dependent and site-specific (Exp. 2). The results are discussed in relation with an earlier report showing that CTA acquisition is disrupted by injection of Ca(2+)/phospholipid-dependent protein kinase (PKC) inhibitor chelerythrine into the PBN during CS-US interval. It is suggested that the principal serine/threonine kinases play different roles in CTA learning: whereas PKC activity is necessary for the gustatory short-term memory formation, CAMKII acts similarly to the US itself-an unexpected role of CAMKII in associative learning.

Role of the perirhinal cortex in rats' conditioned taste aversion response memorization.

The role of the perirhinal cortex (PC) in conditioned taste aversion (CTA) learning was investigated in Long Evans rats. CTA was induced by the intraperitoneal administration of LiCl 60 min after saccharin-sweetened water drinking. The PC was reversibly inactivated by the stereotaxic administration of tetrodotoxin (TTX) 60 min before saccharin drinking, immediately after saccharin drinking (Experiment 1), 6 or 24 hr after LiCl administration (Experiment 2), and 60 min before CTA retrieval testing (Experiment 3). Only pre-saccharin drinking PC inactivation disrupted CTA. Thus, PC integrity is necessary only during the earliest phases of CTA mnemonic processing, that is, taste information acquisition and early gustatory memory elaboration. The results are discussed in relation to PC connectivity and PC temporal involvement in the memorization process of other aversive responses.

Effects of coupled perirhinal cortex and medial septal area, fimbria-fornix, entorhinal cortex tetrodotoxin inactivations on passive avoidance consolidation in the rat.

In order to ascertain the rat perirhinal cortex (PC) function during early consolidation of a passive avoidance response (PAR), and to ascertain whether there are some functional interactions with the medial septal area (MSA), the fimbria-fornix complex (FF) and the entorhinal cortex (EC), PC-MSA, PC-FF, and PC-EC coupled inactivations were performed immediately after the PAR acquisition session. Anesthetized male adult Wistar rats aged 60 days were treated with stereotaxical bilateral injections of TTX (5 ng in 0.5 microl saline) in the appropriate sites. Retrieval testing was performed 48 h later. It was shown that all three coupled inactivations were followed by significant PAR disruption. It may be concluded that PC is somehow active even during the first mnemonic phase following the acquisition session, thus better defining PC mnemonic involvement chronology. These results may be taken as indicating that during initial consolidation the engram is concurrently processed in more than one septal and parahippocampal site, each of which by itself is not absolutely necessary for the final engram formation.

Auditory thalamus, dorsal hippocampus, basolateral amygdala, and perirhinal cortex role in the consolidation of conditioned freezing to context and to acoustic conditioned stimulus in the rat.

On the basis of previous experimental evidence, it is known that the auditory thalamus (AT), the dorsal hippocampus (DH), the basolateral amygdala (BLA), and the perirhinal cortex (PC) are involved in the mnemonic processing of conditioned freezing. In particular, BLA and PC appear to be involved both in conditioned stimulus (CS) and context conditioned freezing. Through AT, the auditory CS is sent to other sites, whereas DH is involved in context conditioning. Nevertheless, the existing evidence does not make it possible to assess AT, DH, BLA, and PC involvement during the consolidation phase of conditioned freezing. To address this question, fully reversible tetrodotoxin (TTX) inactivation was performed on adult male Wistar rats having undergone CS and context fear training. Anesthetized animals were injected stereotaxically with TTX (either 5 or 10 ng in 0.5 or 1.0 microliter of saline, according to site dimensions) at increasing post-acquisition delays. Context and CS freezing durations were measured during retention testing, always performed 48 and 72 hr after TTX administration. The results showed that AT inactivation does not disrupt consolidation of either contextual or auditory fear memories. In contrast, inactivation of the other three structures disrupted consolidation. For the DH, this disruption was specific to contextual cues and only occurred when inactivation was performed early (up to 1.5 hr) after training. The BLA and PC were shown to be involved in the consolidation of both contextual and auditory fear. Their involvement persisted for longer periods of time (2d for BLA and 8 d for PC). These findings provide information to build a temporal profile for the post-training processing of fear memories in structures known to be important for this form of learning. The results are discussed in relation to previous studies on conditioned freezing and other aversive conditioned response neural correlates.

Memorization of contextual and CS conditioned fear response (freezing) in a one-trial acquisition paradigm.

In fear-conditioned Wistar rats freezing was induced by the delivery of a series of footshocks paired to tones (CS) in a specific conditioning chamber (context). CS and contextual fear were acquired in the same single conditioning session without preexposition to the conditioning chamber (day 1). Different groups of animals were conditioned employing three increasing US (footshock) intensities (0.25, 0.5, 0.75 mA). During the retention sessions context and CS conditioned freezing (fear response) were measured using a paradigm that fulfilled the following conditions: i) CS freezing retention was measured in a context different from the conditioning one; ii) CS and context freezing were measured at increased delays after the training session (days 3 and 4, 14 and 15, 28 and 29). The results show that there are significant differences between CS and context freezing retention, which are clearly related to delay after the initial session and to US intensity. In particular: 1) conditioned freezing to a discrete tone is better retained than conditioned freezing to context (irrespective of US intensity); 2) context freezing is directly related to US intensity much more than to tone freezing; 3) context freezing is easier to extinguish than tone freezing. The results are discussed in relation to previous ones and to their relevance to freezing genesis neural correlates.

Effects of combined medial septal area, fimbria-fornix and entorhinal cortex tetrodotoxin inactivations on passive avoidance response consolidation in the rat.

On the basis of previous experimental evidence, it has been concluded that the entorhinal cortex (EC), the fimbria-fornix (FF) complex and medial septal area (MSA) do not take part in the consolidation phase of passive avoidance response (PAR) memorization. On the other hand, a mnemonic role during consolidation of at least two of these structures has been argued, based on several considerations. In order to ascertain whether the EC and FF are still involved in PAR memorization during consolidation, the coupled fully reversible functional tetrodotoxin (TTX) inactivation of MSA, FF and EC was performed in rats having undergone a PAR training. In Experiment 1 MSA, FF and EC were inactivated pair-wise (FF and EC always bilaterally). Permanently cannulated animals were injected stereotaxically with TTX (5 ng in 0.5 microliter saline) or saline (0.5 microliter) immediately following PAR acquisition. It was shown that combined FF-EC inactivation induced PAR retention impairment, whereas FF-MSA and EC-MSA inactivation was not followed by amnesic effects. Having obtained a positive result, in Experiment 2 the combined FF-EC inactivation was performed at different post-acquisition delays (0.25 h, 1.5 h, 6 h), so as to assess the duration of their involvement in PAR consolidation. It was shown that only the coupled inactivation performed at the shortest post-acquisition delay was followed by amnesic effects. Thus EC and FF play a definite role during early consolidation. The results are discussed in relation to EC, FF, MSA, and hippocampal involvement in PAR memorization, as reported in previous studies, and to their connectivity.

Neural topography and chronology of memory consolidation: a review of functional inactivation findings.

Findings on the role of subcortical and cortical structures in mnemonic processes, obtained by means of the reversible functional inactivation technique, are reviewed. The main advantage of this method (subcortical or cortical administration of local anesthetics or tetrodotoxin) is that it provides information not only on "where" but also "when" and for "how long" these processes take place, thus adding to the topographical dimension the chronological one. The review covers several types of memory (e.g., passive avoidance and spatial memory) studies examining the neural substrates of memory consolidation on the basis of the functional inactivation of the nucleus of the solitary tract, parabrachial nuclei, substantia nigra, hippocampus (dorsal and ventral), nucleus basalis magnocellularis, amygdala, medial septal area, striatum, olfactory bulb, and neocortex. The data are discussed in relation to earlier research and with respect to the anatomical and functional connectivity of the examined centers.

A peculiar pattern of temporal involvement of rat perirhinal cortex in memory processing.

By means of the fully reversible tetrodotoxin inactivation technique, perirhinal cortex (PC) mnemonic function was investigated in rats trained to a passive avoidance response (PAR). It was shown that PC functional integrity is necessary during PAR acquisition, during late and very late consolidation (from 24 hr up to 192 hr after the training session), and during retrieval. An unexpected finding was that the PC was not involved in the early consolidation period. Thus the PC may play a relatively simple relay or connective role during acquisition, but its very late and very long consolidative involvement may indicate a peculiar function in consolidation and possibly in the storage of the PAR engram. The results are discussed in terms of the mnemonic characteristics of other neural sites (amygdala, hippocampus, and entorhinal cortex) involved in the same learning process.

Temporal characterization of subcortical nuclei in mnemonic processes: results of tetrodotoxin reversible inactivation studies in the rat.

By means of permanent lesion techniques it has been possible to ascertain whether a given subcortical neural structure is involved in memory processing. These results, however, are useful only to build a topography of memory, i.e. to provide information only on the "where" such processes take place. Memory being, per se, a temporal process, organized in at least three putative phases (acquisition, consolidation, retrieval) it is of paramount importance to know not only the "where", but also the "when", and, possibly, the "how long" of a given site involvement. The fully reversible inactivation technique has been employed to assess the chronological involvement of subcortical sites. By means of the stereotaxic administration of tetrodotoxin (TTX) it has been possible to inactivate known volumes of nervous tissue for given periods of time. In this way, it has been possible to measure the amnesic effects (disruption of the performance of a passive avoidance response, PAR) after inactivation of discrete neural sites. The data so far obtained by these means are presented and discussed. The comparison of results is justified by the constancy of the experimental subjects (young adult male rats of the same age), the surgical interventions, and the conditioning paradigm (passive avoidance responding in the light-dark box). The parabrachial nuclei, substantia nigra, ventral hippocampus, dorsal hippocampus, nucleus basalis magnocellularis, amygdala, globus pallidus, nucleus caudate-putamen (anterior, median, posterior), medial septal area and nucleus accumbens have been investigated. From these studies, data have been acquired on all three phases of memorization. The most detailed findings concern consolidation. In particular, it was shown that the functional integrity duration necessary to avoid amnesic damages varies greatly from site to site, from at least 15 min to no less than 48 hours. The results confirm and amplify previous experimental work, by defining the chronology of mnemonic involvement of many neural sites. The results are discussed in terms of comparison between sites and connectivity between the investigated sites and other sites or neuronal systems.