The use of mantrailing dogs in police and judicial context, future directions, limits and possibilities - A law review.

The extraordinary capabilities of the canine nose are increasingly being used by law enforcement agencies in many countries to solve and reconstruct crimes. As a result, this type of forensic evidence can be and is still being challenged in the courts. So far, only a few publications have addressed the jurisprudence concerning mantrailing. We provide an overview of the jurisprudence in Germany and the USA, as well as insights from France. Relevant databases were searched, and 201 verdicts from Germany and 801 verdicts from the USA were analyzed. As a result, 16 published verdicts on the topic of mantrailing were found for Germany, and 44 verdicts since 2010 were found for the USA. The use of mantrailers and human scent discrimination dogs is employed in the investigative process in all three countries. The results derived from these methods are admissible as evidence in court, albeit not as sole evidence.

Improving Stereotaxic Neurosurgery Techniques and Procedures Greatly Reduces the Number of Rats Used per Experimental Group-A Practice Report.

Techniques of stereotaxic surgery are commonly used in research laboratories by a range of students, technicians, and researchers. To meet the evolving requirements imposed by international legislation, and to promote the implementation of 3R rules (replacement, reduction, and refinement) by reducing experimental error, animal morbidity, and mortality, it is essential that standard operating procedures and proper conduct following such complex surgeries be precisely described and respected. The present report shows how refinements of our own neurosurgical techniques over decades, have significantly reduced the number of animals (rats) used in experiments and improved the animals' well-being during the post-surgical recovery period. The current pre-, per-, and post-surgical procedures used in our laboratory are detailed. We describe the practical aspects of stereotaxic neurosurgery that have been refined in our laboratory since 1992 and that cover various areas including appropriate anesthesia and pain management during and after surgery, methods to determine the stereotaxic coordinates, and the best approach to the target brain structure. The application of these optimal surgical methods that combine reliable and reproducible results with an acute awareness of ethics and animal welfare leads to a significant reduction in the number of animals included in experimental research in accordance with ethical and regulatory rules as required by the European Directive on laboratory animal welfare.

Hyperexcitability and seizures in the THY-Tau22 mouse model of tauopathy.

Epileptic seizures constitute a significant comorbidity of Alzheimer's disease (AD), which are recapitulated in transgenic mouse models of amyloidogenesis. Here, we sought to evaluate the potential role of tau pathology regarding seizure occurrence. To this end, we performed intra-hippocampal electroencephalogram (EEG) recordings and PTZ (pentylenetetrazol) seizure threshold tests in THY-Tau22 transgenic mice of AD-like tau pathology. We demonstrate that despite a lack of spontaneous epileptiform activity in Tau22 mice, the animals display increased PTZ-induced seizure susceptibility and mortality. The increased propensity for induced seizures in THY-Tau22 mutants correlates with astrogliosis and increased expression of adenosine kinase, consistent with increased network excitability. These data support an impact of tau pathology toward AD-associated seizures and suggest that tau pathology may contribute to seizure generation in AD independent of Aß pathology.

Scent lineups compared across eleven countries: Looking for the future of a controversial forensic technique.

A scent lineup is generally a procedure whereby a dog's alerting behavior is used to establish that the dog detects two scents, one from a crime scene and one from a suspect, as deriving from the same person. The aim of this article is to compare methodologies of using dogs in scent lineups as a means of identifying perpetrators of crimes. It is hoped that this comparative approach, looking at countries where the method is currently or has in the past been used, will help determine what issues should be addressed in order to assure that the scent lineup will have a future as a forensic technique. Participants from eleven countries-Belgium, The Czech Republic, Finland, France, Germany, Hungary, Lithuania, The Netherlands, Poland, Russia, and the U.S.-completed a survey questionnaire regarding key aspects of the scent lineup procedures used by the police in their countries. Although there was broad overlap on certain matters, such as the use of control and zero trials, collection of decoy scents from individuals of similar gender and race as the suspect, materials for holding scent, frequency of cleaning and changing stations, and use and timing of rewards, there were significant differences in the degree of blindness required, who calls an alert (handler or experimenter), and whether handlers can work with more than one dog. The gap between recommendations and results available from the scientific literature and procedures used in police practice was greater for some countries than others, even taking into account that some scientific methodologies might be expensive or impractical given agency resources. The authors make recommendations about how to go forward if scent lineups are to remain a valid forensic technique.

Microdialysis Unveils the Role of the α2-Adrenergic System in the Basolateral Amygdala during Acquisition of Conditioned Odor Aversion in the Rat.

Previous work has shown that ß-adrenergic and GABAergic systems in the basolateral amygdala (BLA) are involved in the acquisition of conditioned odor aversion (COA) learning. The involvement of α2-adrenoreceptors, however, is poorly documented. In a first experiment, male Long-Evans rats received infusions of 0.1 µg of the selective α2-antagonist dexefaroxan (Dex) in the BLA before being exposed to COA learning. In a second experiment, levels of norepinephrine (NE) were analyzed following Dex retrodialysis into the BLA. While microdialysis data showed a significant enhancement of NE release in the BLA with Dex, behavioral results showed that pre-CS infusion of Dex impaired, rather than facilitated, the acquisition of COA. Our results show that the NE system in the BLA is involved in the acquisition of COA, including a strong α2-receptor modulation until now unsuspected. Supported by the recent literature, the present data suggest moreover that the processes underlying this learning are probably mediated by the balanced effects of NE excitatory/inhibitory signaling in the BLA, in which interneurons are highly involved.

Influence of early stress on memory reconsolidation: Implications for post-traumatic stress disorder treatment.

Post-traumatic stress disorder (PTSD) is a common consequence of exposure to a life-threatening event. Currently, pharmacological treatments are limited by high rates of relapse, and novel treatment approaches are needed. We have recently demonstrated that propranolol, a ß-adrenergic antagonist, inhibited aversive memory reconsolidation in animals. Following this, in an open-label study 70% of patients with PTSD treated with propranolol during reactivation of traumatic memory exhibited full remission. However, the reason why 30% of these patients did not respond positively to propranolol treatment is still unclear. One of the major candidates as factor of treatment resistance is the patient's early-life traumatic history. To test the role of this factor, mice with pre- or postnatal stress are being tested in fear conditioning and in a new behavioral task, the "city-like", specifically designed as a mouse model of PTSD. After reactivation of the traumatic event, mice received propranolol injection to block the noradrenergic system during memory reconsolidation. Results show that, in the "city-like" test, control mice strongly avoided the shock compartment but also the compartments containing cues associated with the electric shocks. Injection of propranolol after reactivation greatly reduced the memory of the traumatic event, but this effect was not present when mice had received pre- or postnatal stress. Moreover, propranolol produced only a very weak effect in the fear conditioning test, and never changed the corticosterone level whatever the behavioral experiment. Taken together our results suggest that our new behavioural paradigm is well adapted to PTSD study in mice, and that early stress exposure may have an impact on propranolol PTSD treatment outcome. These data are critical to understanding the effect of propranolol treatment, in order to improve the therapeutic protocol currently used in humans.

Neuronal dynamics supporting formation and recombination of cross-modal olfactory-tactile association in the rat hippocampal formation.

The present study is aimed at describing some aspects of the neural dynamics supporting discrimination of olfactory-tactile paired-associated stimuli during acquisition of new pairs and during recombination of previously learned pairs in the rat. To solve the task, animals have to identify one odor-texture (OT) combination associated with a food reward among three cups with overlapping elements. Previous experiments demonstrated that the lateral entorhinal cortex (LEC) is involved in the processes underlying OT acquisition, whereas the dorsal hippocampus (DH) is selectively involved in the recombination processes. In the present study, local field potentials were recorded form the anterior piriform cortex (aPC), LEC, and DH in freely moving rats performing these tasks. Signal analysis focused on theta (5-12 Hz)- and beta-band (15-40 Hz) oscillatory activities in terms of both amplitude and synchrony. The results show that cue sampling was associated with a significant increase in the beta-band activity during the choice period in both the aPC and the LEC, and is modulated by level of expertise and the animal's decision. In addition, this increase was significantly higher during the recombination compared with the acquisition of the OT task, specifically when animals had to neglect the odor previously associated with the reward. Finally, a significant decrease in coherence in the theta band between LEC and DH was observed in the recombination but not in the acquisition task. These data point to specific neural signatures of simple and complex cross-modal sensory processing in the LEC-DH complex. NEW & NOTEWORTHY This study is the first to describe electrophysiological correlates of cross-modal olfactory-tactile integration in rats. Recordings were sought from the lateral entorhinal cortex and the dorsal hippocampus because previous studies have shown their role in the formation and in the recombination of previously learned associations. We identified specific oscillatory-evoked neural responses in these structures in the theta and beta bands, which characterize acquisition and recombination of cross-modal olfactory-tactile pairs.

Respective role of the dorsal hippocampus and the entorhinal cortex during the recombination of previously learned olfactory-tactile associations in the rat.

The hippocampal formation has been extensively described as a key component for object recognition in conjunction with place and context. The present study aimed at describing neural mechanisms in the hippocampal formation that support olfactory-tactile (OT) object discrimination in a task where space and context were not taken into account. The task consisted in discriminating one baited cup among three, each of them presenting overlapping olfactory or tactile elements. The experiment tested the involvement of the entorhinal cortex (EC) and the dorsal hippocampus (DH) in the acquisition of this cross-modal task, either with new items or with familiar but recombined items. The main results showed that DH inactivation or cholinergic muscarinic blockade in the DH selectively and drastically disrupted performance in the recombination task. EC inactivation impaired OT acquisition of any OT combinations while cholinergic blockade only delayed it. Control experiments showed that neither DH nor EC inactivation impaired unimodal olfactory or tactile tasks. As a whole, these data suggest that DH-EC interactions are of importance for flexibility of cross-modal representations with overlapping elements.

Rigorous Training of Dogs Leads to High Accuracy in Human Scent Matching-To-Sample Performance.

Human scent identification is based on a matching-to-sample task in which trained dogs are required to compare a scent sample collected from an object found at a crime scene to that of a suspect. Based on dogs' greater olfactory ability to detect and process odours, this method has been used in forensic investigations to identify the odour of a suspect at a crime scene. The excellent reliability and reproducibility of the method largely depend on rigor in dog training. The present study describes the various steps of training that lead to high sensitivity scores, with dogs matching samples with 90% efficiency when the complexity of the scents presented during the task in the sample is similar to that presented in the in lineups, and specificity reaching a ceiling, with no false alarms in human scent matching-to-sample tasks. This high level of accuracy ensures reliable results in judicial human scent identification tests. Also, our data should convince law enforcement authorities to use these results as official forensic evidence when dogs are trained appropriately.

The entorhinal cortex is involved in conditioned odor and context aversions.

In a natural environment, avoidance of a particular food source is mostly determined by a previous intake experience during which sensory stimuli such as food odor, become aversive through a simple associative conditioned learning. Conditioned odor aversion learning (COA) is a food conditioning paradigm that results from the association between a tasteless scented solution (conditioned stimulus, CS) and a gastric malaise (unconditioned stimulus, US) that followed its ingestion. In the present experimental conditions, acquisition of COA also led to acquisition of aversion toward the context in which the CS was presented (conditioned context aversion, CCA). Previous data have shown that the entorhinal cortex (EC) is involved in the memory processes underlying COA acquisition and context fear conditioning, but whether EC lesion modulates CCA acquisition has never be investigated. To this aim, male Long-Evans rats with bilateral EC lesion received CS-US pairings in a particular context with different interstimulus intervals (ISI). The results showed that the establishment of COA with long ISI obtained in EC-lesioned rats is associated with altered CCA learning. Since ISI has been suggested to be the determining factor in the odor- and context-US association, our results show that the EC is involved in the processes that control both associations relative to ISI duration.

Noradrenergic influences in the basolateral amygdala on inhibitory avoidance memory are mediated by an action on α2-adrenoceptors.

The role of norepinephrine (NE) in the consolidation of inhibitory avoidance learning (IA) in rats is known to involve α1- and ß-adrenoceptor systems in the basolateral nucleus of the amygdala (BLA). However, the amygdala also contains α2-adrenoceptor subtypes, and local microinfusions of the selective α2-adrenoceptor antagonist idazoxan and agonist UK 14,304 respectively into the BLA enhance and inhibit IA performances when administered before acquisition. The present study investigated whether the effects of idazoxan and UK 14,304 on IA were associated with changes in NE release within the BLA before and after one-trial inhibitory avoidance training. Male Sprague-Dawley rats were unilaterally implanted with a microdialysis probe in the BLA and were administered idazoxan (0.1mM) or UK 14,304 (10 µM) by retrodialysis infusion 15 min before the acquisition of IA. Dialysates were collected every 15 min for analysis of NE. Retrodialysis of idazoxan potentiated the release of NE induced by footshock application, whereas UK 14,304 decreased NE release to the extent that the footshock failed to induce any measurable effect on NE levels. Idazoxan infusion enhanced IA retention tested 24h later and this effect was directly related to the level of NE release in the BLA measured during IA acquisition. In contrast, the infusion of UK 14,304 did not modify IA performances in comparison to control animals, possibly due to compensatory activity of the contralateral BLA. These results are consistent with previous evidence that amygdala NE is involved in modulating memory consolidation, and provide evidence for an involvement of presynaptic α2-autoceptors in the BLA in this process.

The orexinergic system influences conditioned odor aversion learning in the rat: a theory on the processes and hypothesis on the circuit involved.

A large variety of behaviors that are essential for animal survival depend on the perception and processing of surrounding smells present in the natural environment. In particular, food-search behavior, which is conditioned by hunger, is directly driven by the perception of odors associated with food, and feeding status modulates olfactory sensitivity. The orexinergic hypothalamic peptide orexin A (OXA), one of the central and peripheral hormones that triggers food intake, has been shown to increase olfactory sensitivity in various experimental conditions including the conditioned odor aversion learning paradigm (COA). COA is an associative task that corresponds to the association between an olfactory conditioned stimulus (CS) and a delayed gastric malaise. Previous studies have shown that this association is formed only if the delay separating the CS presentation from the malaise is short, suggesting that the memory trace of the odor is relatively unstable. To test the selectivity of the OXA system in olfactory sensitivity, a recent study compared the effects of fasting and of central infusion of OXA during the acquisition of COA. Results showed that the increased olfactory sensitivity induced by fasting and by OXA infusion was accompanied by enhanced COA learning performances. In reference to the duration of action of OXA, the present work details the results obtained during the successive COA extinction tests and suggests a hypothesis concerning the role of the OXA component of fasting on the memory processes underlying CS-malaise association during COA. Moreover, referring to previous data in the literature we suggest a functional circuit model where fasting modulates olfactory memory processes through direct and/or indirect activation of particular OXA brain targets including the olfactory bulb, the locus coeruleus (LC) and the amygdala.

Involvement of the lateral entorhinal cortex for the formation of cross-modal olfactory-tactile associations in the rat.

While the olfactory and tactile vibrissal systems have been extensively studied in the rat, the neural basis of these cross-modal associations is still elusive. Here we tested the hypothesis that the lateral entorhinal cortex (LEC) could be particularly involved. In order to tackle this question, we have developed a new behavioral paradigm which consists in finding one baited cup (+) among three, each of the cups presenting a different and specific odor/texture (OT) combination. During the acquisition of a first task (Task OT1), the three cups were associated with the following OT combination: O1T1 for the baited cup; O2T1 and O1T2 for non-baited ones. Most rats learn this task within three training sessions (20 trials/session). In a second task (Task OT2) animals had to pair another OT combination with the reward using a new set of stimuli (O3T3+, O4T3, and O3T4). Results showed that rats manage to learn Task OT2 within one session only. In a third task (Task OT3) animals had to learn another OT combination based on previously learned items (e.g. O4T4+, O1T4 and O4T1). This task is called the "recombination task." Results showed that control rats solve the recombination task within one session. Animals bilaterally implanted with cannulae in the LEC were microinfused with d-APV (3 µg/0.6 µL) just before the acquisition or the test session of each task. The results showed that NMDA receptor blockade in LEC did not affect recall of Task OT1 but strongly impaired acquisition of both Task OT2 and OT3. Moreover, two control groups of animals infused with d-APV showed no deficit in the acquisition of unimodal olfactory and tactile tasks. Taken together, these data show that the NMDA system in the LEC is involved in the acquisition of association between an olfactory and a tactile stimulus during cross-modal learning task.

The orexin component of fasting triggers memory processes underlying conditioned food selection in the rat.

To test the selectivity of the orexin A (OXA) system in olfactory sensitivity, the present study compared the effects of fasting and of central infusion of OXA on the memory processes underlying odor-malaise association during the conditioned odor aversion (COA) paradigm. Animals implanted with a cannula in the left ventricle received ICV infusion of OXA or artificial cerebrospinal fluid (ACSF) 1 h before COA acquisition. An additional group of intact rats were food-deprived for 24 h before acquisition. Results showed that the increased olfactory sensitivity induced by fasting and by OXA infusion was accompanied by enhanced COA performance. The present results suggest that fasting-induced central OXA release influenced COA learning by increasing not only olfactory sensitivity, but also the memory processes underlying the odor-malaise association.

Analysis of microdialysate monoamines, including noradrenaline, dopamine and serotonin, using capillary ultra-high performance liquid chromatography and electrochemical detection.

Electrochemical methods are very often used to detect catecholamine and indolamine neurotransmitters separated by conventional reverse-phase high performance liquid chromatography (HPLC). The present paper presents the development of a chromatographic method to detect monoamines present in low-volume brain dialysis samples using a capillary column filled with sub-2µm particles. Several parameters (repeatability, linearity, accuracy, limit of detection) for this new ultrahigh performance liquid chromatography (UHPLC) method with electrochemical detection were examined after optimization of the analytical conditions. Noradrenaline, adrenaline, serotonin, dopamine and its metabolite 3-methoxytyramine were separated in 1µL of injected sample volume; they were detected above concentrations of 0.5-1nmol/L, with 2.1-9.5% accuracy and intra-assay repeatability equal to or less than 6%. The final method was applied to very low volume dialysates from rat brain containing monoamine traces. The study demonstrates that capillary UHPLC with electrochemical detection is suitable for monitoring dialysate monoamines collected at high sampling rate.

Involvement of basolateral amygdala alpha2-adrenoceptors in modulating consolidation of inhibitory avoidance memory.

These experiments investigated the role of the alpha(2)-adrenoceptors of the basolateral nucleus of the amygdala (BLA) in modulating the retention of inhibitory avoidance (IA). In Experiment 1, male Sprague Dawley rats implanted with bilateral cannulae in the BLA received microinfusions of a selective alpha(2)-adrenoceptor antagonist idazoxan 20 min either before or immediately after training. Retention was tested 48 h later. Idazoxan induced a dose-dependent enhancement of retention performance and was more effective when administered post-training. In Experiment 2, animals received pre- or post-training intra-BLA infusions of a selective alpha(2)-adrenoceptor agonist UK 14,304. The agonist induced a dose-dependent impairment of retention performance and, as with the antagonist treatments, post-training infusions were more effective. These results provide additional evidence that consolidation of inhibitory avoidance memory depends critically on prolonged activation of the noradrenergic system in the BLA and indicate that this modulatory influence is mediated, in part, by pre-synaptic alpha(2)-adrenoceptors.

Differential effects of beta-adrenergic receptor blockade in basolateral amygdala or insular cortex on incidental and associative taste learning.

The importance of central beta-adrenergic system has been essentially investigated in aversive/emotional learning tasks. However, recent data suggest that the beta-adrenergic system is also required for incidental taste learning. In the present study we evaluated in rats whether beta-adrenergic receptor activity is required for taste habituation, an incidental taste learning, and also for conditioned taste aversion (CTA) learning, an associative learning. To address this issue, a low dose of the beta-adrenergic antagonist propranolol was infused before learning in either the basolateral amygdala (BLA) or the insular cortex (IC), two forebrain areas reported to play a key role in taste memory formation. Incidental taste learning was assessed using a single presentation of the sweet taste saccharin 0.1%, which is sufficient to increase saccharin consumption (relative to water baseline) during a second presentation. CTA was assessed by pairing the first saccharin 0.1% presentation with a delayed gastric malaise, thus causing a decrease in saccharin consumption (relative to water baseline) during a second presentation. Propranolol infusion in BLA (1microg/0.2microl) or IC (2.5microg/0.5microl) before the first taste exposure impaired incidental taste learning but did not affect CTA. These results highlight the important role played by the beta-adrenergic receptor activation in cortical and amygdaloid structures during taste learning. Moreover, they are the first to suggest that incidental learning is more sensitive to blockade of noradrenergic system than associative learning.

Basolateral amygdala noradrenergic activity is involved in the acquisition of conditioned odor aversion in the rat.

Conditioned odor aversion (COA) is the avoidance of an odorized-tasteless solution (the conditioned stimulus, CS), the ingestion of which precedes toxicosis. Previous works have shown that the basolateral nucleus of the amygdala (BLA) is involved in the acquisition, and more precisely, the control of the CS memory trace, of COA. Since catecholamine depletion of the amygdala induced a deficit in the potentiated version of COA, this study investigated the role of the adrenergic system in the BLA during COA. Male Wistar rats bilaterally implanted with cannulae aimed at the BLA were microinjected with the beta-adrenergic antagonist propranolol (1 microg/0.2 microl) during the acquisition (5 min before the CS presentation, pre-CS, or immediately after, post-CS) or during the retrieval test (5 min before test, pre-test). Results showed that pre-CS, but neither post-CS nor pre-test, infusions of propranolol impaired COA, suggesting that beta-adrenergic system activity in the BLA is involved in the acquisition but not the expression of COA. Moreover, the fact that pre-CS, but not post-CS, treatment disrupted COA suggests that beta-adrenergic system in the BLA is involved in the initiation but not the maintenance of the CS memory trace during COA acquisition.

Immunotoxic cholinergic lesions in the basal forebrain reverse the effects of entorhinal cortex lesions on conditioned odor aversion in the rat.

Conditioned odor aversion (COA) corresponds to the avoidance of an odorized-tasteless solution (conditioned stimulus, CS) previously paired with toxicosis. COA occurs only when the interstimulus interval (ISI) is kept short, suggesting that the memory trace of the odor is subject to rapid decay. Previous experiments have shown that the entorhinal cortex (EC) is involved in the acquisition of COA, since lesion of the EC rendered COA tolerant to long ISI. Because EC lesions induce a septo-hippocampal cholinergic sprouting, the present experiment investigated whether COA tolerance to long ISI may be linked to this sprouting reaction. In a first experiment, male Long-Evans rats subjected to bilateral excitotoxic EC lesions combined to intracerebroventricular infusions of the selective cholinergic immunotoxin 192 IgG-saporin were exposed to odor-toxicosis pairing using a long ISI (120 min). Results showed that EC-lesioned rats displayed COA with the long ISI but not the control groups. In rats with EC combined to 192 IgG-saporin lesions, histological analysis demonstrated no evidence for cholinergic septo-hippocampal sprouting. In a second experiment, animals with 192-IgG saporin lesion showed a marked COA with a short ISI (5 min). These results suggest that the COA with the long ISI found in rats with EC lesions might involve a functional activity related to the EC lesion-induced hippocampal cholinergic sprouting. As the injection of 192 IgG-saporin alone did not affect COA with a short ISI, our data also point to a possible role of hippocampal cholinergic neurons in the modulation of memory processes underlying COA.

Combined damage to entorhinal cortex and cholinergic basal forebrain neurons, two early neurodegenerative features accompanying Alzheimer's disease: effects on locomotor activity and memory functions in rats.

In Alzheimer's disease (AD), cognitive decline is linked to cholinergic dysfunctions in the basal forebrain (BF), although the earliest neuronal damage is described in the entorhinal cortex (EC). In rats, selective cholinergic BF lesions or fiber-sparing EC lesions may induce memory deficits, but most often of weak magnitude. This study investigated, in adult rats, the effects on activity and memory of both lesions, alone or in combination, using 192 IgG-saporin (OX7-saporin as a control) and L-N-methyl-D-aspartate to destroy BF and EC neurons, respectively. Rats were tested for locomotor activity in their home cage and for working- and/or reference-memory in various tasks (water maze, Hebb-Williams maze, radial maze). Only rats with combined lesions showed diurnal and nocturnal hyperactivity. EC lesions impaired working memory and induced anterograde memory deficits in almost all tasks. Lesions of BF cholinergic neurons induced more limited deficits: reference memory was impaired in the probe trial of the water-maze task and in the radial maze. When both lesions were combined, performance never improved in the water maze and the number of errors in the Hebb-Williams and the radial mazes was always larger than in any other group. These results (i) indicate synergistic implications of BF and EC in memory function, (ii) suggest that combined BF cholinergic and fiber-sparing EC lesions may model aspects of anterograde memory deficits and restlessness as seen in AD, (iii) challenge the cholinergic hypothesis of cognitive dysfunctions in AD, and (iv) contribute to open theoretical views on AD-related memory dysfunctions going beyond the latter hypothesis.