A new model of experimental hemispherotomy in young adult Rattus norvegicus: a neural tract tracing and SPECT in vivo study.

OBJECTIVE: The objective of this study was to describe a new experimental model of hemispherotomy performed on laboratory animals. METHODS: Twenty-six male young adult Wistar rats were distributed into two groups (surgery and control). The nonfluorescent anterograde neurotracer biotinylated dextran amine (BDA; 10,000 MW) was microinjected into the motor cortex area (M1) according to The Rat Brain in Stereotaxic Coordinates atlas to identify pathways and fibers disconnected after the experimental hemispherectomy. SPECT tomographic images of 99mTc hexamethylpropyleneamine oxime were obtained to verify perfusion in functioning areas of the disconnected and intact brain. A reproducible and validated surgical procedure is described in detail, including exact measurements and anatomical relationships. An additional 30 rodents (n = 10 rats per group) were divided into naïve, sham, and hemispherotomy groups and underwent the rotarod test. RESULTS: Cortico-cortical neural pathways were identified crossing the midline and contacting neuronal perikarya in the contralateral brain hemisphere in controls, but not in animals undergoing hemispherotomy. There was an absence of perfusion in the left side of the brain of the animals undergoing hemispherotomy. Motor performance was significantly affected by brain injuries, increasing the number of attempts to maintain balance on the moving cylinder in the rotarod test at 10 and 30 days after the hemispherotomy, with a tendency to minimize the motor performance deficit over time. CONCLUSIONS: The present findings show that the technique reproduced neural disconnection with minimal resection of brain parenchyma in young adult rats, thereby duplicating the hemispherotomy procedures in human patients.

Innate defensive behaviour and panic-like reactions evoked by rodents during aggressive encounters with Brazilian constrictor snakes in a complex labyrinth: behavioural validation of a new model to study affective and agonistic reactions in a prey versus predator paradigm.

Defensive behaviour has been extensively studied, and non-invasive methodologies may be interesting approaches to analyzing the limbic system function as a whole. Using experimental models of animals in the state of anxiety has been fundamental in the search for new anxiolytic and antipanic compounds. The aim of this present work is to examine a new model for the study of affective behaviour, using a complex labyrinth consisting of an arena and galleries forming a maze. Furthermore, it aims to compare the defensive behaviour of Wistar rats, Mongolian gerbils and golden hamsters in a complex labyrinth, as well as the defensive behaviour of Meriones unguiculatus in aggressive encounters with either Epicrates cenchria assisi or Boa constrictor amarali in this same model. Among species presently studied, the Mongolian gerbils showed better performance in the exploration of both arena and galleries of the labyrinth, also demonstrating less latency in finding exits of the galleries. This increases the possibility of survival, as well as optimizes the events of encounter with the predator. The duration of alertness and freezing increased during confrontation with living Epicrates, as well as the duration of exploratory behaviour in the labyrinth. There was an increase in the number of freezing and alertness behaviours, as well as in duration of alertness during confrontations involving E.c. assisi, compared with behavioural reactions elicited by jirds in presence of B.c. amarali. Interestingly, the aggressive behaviour of Mongolian gerbils was more prominent against B.c. amarali compared with the other Boidae snake. E.c. assisi elicited more offensive attacks and exhibited a greater time period of body movement than B.c. amarali, which spent more time in the arena and in defensive immobility than the E.c. assisi. Considering that jirds evoked more fear-like reaction in contact with E.c. assisi, a fixed E.c. assisi kept in a hermetically closed acrylic box was used as control. In these prey/predator encounter-based experiments, there was an increase in the number of alertness and freezing behaviours exhibited by gerbils, and a decrease in the number of crossing elicited by them, when comparing confrontations between the living E.c. assisi and the control. The experiments were performed at 7.0 p.m. In the labyrinth, the snakes showed in confrontation similar performance to that observed in nature (organizing hunting behaviour, offensive/defensive attack, constriction, prey inspection and feeding behaviour), which were essential to the validity of the experiments and gave behavioural validation within the complex labyrinth.

Unravelling cortico-hypothalamic pathways regulating unconditioned fear-induced antinociception and defensive behaviours.

The medial prefrontal cortex can influence unconditioned fear-induced defensive mechanisms organised by diencephalic neurons that are under tonic GABAergic inhibition. The posterior hypothalamus (PH) is involved with anxiety- and panic attack-like responses. To understand this cortical mediation, our study characterised anterior cingulate cortex (ACC)-PH pathways and investigated the effect of ACC local inactivation with lidocaine. We also investigated the involvement of PH ionotropic glutamate receptors in the defensive behaviours and fear-induced antinociception by microinjecting NBQX (an AMPA/kainate receptor antagonist) and LY235959 (a NMDA receptor antagonist) into the PH. ACC pretreatment with lidocaine decreased the proaversive effect and antinociception evoked by GABAA receptor blockade in the PH, which suggests that there may be descending excitatory pathways from this cortical region to the PH. Microinjections of both NBQX and LY235959 into the PH also attenuated defensive and antinociceptive responses. This suggests that the blockade of AMPA/kainate and NMDA receptors reduces the activity of glutamatergic efferent pathways. Both inputs from the ACC to the PH and glutamatergic hypothalamic short links disinhibited by intra-hypothalamic GABAA receptors blockade are potentially implicated. Microinjection of a bidirectional neurotracer in the PH showed a Cg1-PH pathway and PH neuronal reciprocal connections with the periaqueductal grey matter. Microinjections of an antegrade neurotracer into the Cg1 showed axonal fibres and glutamatergic vesicle-immunoreactive terminal boutons surrounding both mediorostral-lateroposterior thalamic nucleus and PH neuronal perikarya. These data suggest a critical role played by ACC-PH glutamatergic pathways and AMPA/kainate and NMDA receptors in the panic attack-like reactions and antinociception organised by PH neurons.

Opioid neurotransmission modulates defensive behavior and fear-induced antinociception in dangerous environments.

The effects of endogenous opioid peptide antagonists on panic-related responses are controversial. Using elevated mazes and a prey-versus-predator paradigm, we investigated the involvement of the endogenous opioid peptide-mediated system in the modulation of anxiety- and panic attack-induced responses and innate fear-induced antinociception in the present work. Wistar rats were intraperitoneally pretreated with either physiological saline or naloxone at different doses and were subjected to either the elevated plus- or T-maze test or confronted by Crotalus durissus terrificus. The defensive behaviors of the rats were recorded in the presence of the predator and at 24h after the confrontation, when the animals were placed in the experimental enclosure without the rattlesnake. The peripheral non-specific blockade of opioid receptors had a clear anxiolytic-like effect on the rats subjected to the elevated plus-maze but not on those subjected to the elevated T-maze; however, a clear panicolytic-like effect was observed, i.e., the defensive behaviors decreased, and the prey-versus-predator interaction responses evoked by the presence of the rattlesnakes increased. A similar effect was noted when the rats were exposed to the experimental context in the absence of the venomous snake. After completing all tests, the naloxone-treated groups exhibited less anxiety/fear-induced antinociception than the control group, as measured by the tail-flick test. These findings demonstrate the anxiolytic and panicolytic-like effects of opioid receptor blockade. In addition, the fearlessness behavior displayed by preys treated with naloxone at higher doses enhanced the defensive behavioral responses of venomous snakes.

Connexions between the dorsomedial division of the ventromedial hypothalamus and the dorsal periaqueductal grey matter are critical in the elaboration of hypothalamically mediated panic-like behaviour.

The electrical and chemical stimulation of the dorsal periaqueductal grey matter (dPAG) elicits panic-like explosive escape behaviour. Although neurons of the ventromedial hypothalamus (VMH) seem to organise oriented escape behaviour, when stimulated with excitatory amino acids at higher doses, non-oriented/explosive escape reactions can also be displayed. The aim of this work was to examine the importance of reciprocal projections between the VMH and the dPAG for the organisation of this panic-like behaviour. The chemical stimulation of the VMH with 9nmol of N-methyl-d-aspartic acid (NMDA) elicited oriented and non-oriented escape behaviours. The pretreatment of the dPAG with a non-selective blocker of synaptic contacts, cobalt chloride (CoCl2), followed by stimulation of the dorsomedial part of the ventromedial hypothalamus (dmVMH) with 9nmol of NMDA, abolished the non-oriented/explosive escape and freezing responses elicited by the stimulation of the dmVMH. Nonetheless, the rats still showed oriented escape to the burrow. On the other hand, when the blockade of the dmVMH with CoCl2 was followed by stimulation of the dPAG with 6nmol of NMDA, no effect was observed either on the non-oriented/explosive escape or on the freezing behaviour organised by the dPAG. Furthermore, Fos protein-labelled neurons were observed in the dPAG after the stimulation of the dmVMH with 9nmol of NMDA. Additionally, when the anterograde neurotracer biotinylated dextran amine (BDA) was deposited in the dmVMH subsequent stimulation of the dmVMH produced BDA-labelled neural fibres with terminal boutons surrounding Fos-labelled neurons in the dPAG, suggesting synaptic contacts between dmVMH and dPAG neurons for eliciting panic-like behavioural responses. The current data suggest that the dPAG is the key structure that organises non-oriented/explosive escape reactions associated with panic attack-like behaviours.

Descriptive and functional neuroanatomy of locus coeruleus-noradrenaline-containing neurons involvement in bradykinin-induced antinociception on principal sensory trigeminal nucleus.

The present study was carried out in Wistar rats, using the jaw-opening reflex and dental pulp stimulation, to investigate noradrenaline- and serotonin-mediated antinociceptive circuits. The effects of microinjections of bradykinin into the principal sensory trigeminal nucleus (PSTN) before and after neurochemical lesions of the locus coeruleus noradrenergic neurons were studied. Neuroanatomical experiments showed evidence for reciprocal neuronal pathways connecting the locus coeruleus (LC) to trigeminal sensory nuclei and linking monoaminergic nuclei of the pain inhibitory system to spinal trigeminal nucleus (STN). Fast blue (FB) injections in the locus coeruleus/subcoeruleus region retrogradely labeled neurons in the contralateral PSTN and LC. Microinjections of FB into the STN showed neurons labeled in both ipsilateral and contralateral LC, as well as in the ipsilateral Barrington's nucleus and subcoeruleus area. Retrograde tract-tracing with FB also showed that the mesencephalic trigeminal nucleus sends neural pathways towards the ipsilateral PSTN, with outputs from cranial and caudal aspects of the brainstem. In addition, neurons from the lateral and dorsolateral columns of periaqueductal gray matter also send outputs to the ipsilateral PSTN. Microinjections of FB in the interpolar and caudal divisions of the STN labeled neurons in the caudal subdivision of STN. Microinjections in the STN interpolar and caudal divisions also retrogradely labeled serotonin- and noradrenaline-containing nucleus of the brainstem pain inhibitory system. Finally, the gigantocellularis complex (nucleus reticularis gigantocellularis/paragigantocellularis), nucleus raphe magnus and nucleus raphe pallidus also projected to the caudal divisions of the STN. Microinjections of bradykinin in the PSTN caused a statistically significant long-lasting antinociception, antagonized by the damage of locus coeruleus-noradrenergic neuronal fibres with (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) (DSP4), a neurotoxin that specifically depleted noradrenaline from locus coeruleus terminal fields. These data suggest that serotonin- and noradrenaline-containing nuclei of the endogenous pain inhibitory system exert a key-role in the antinociceptive mechanisms of bradykinin and the locus coeruleus is crucially involved in this effect.

Role of muscarinic and nicotinic cholinergic receptors in an experimental model of epilepsy-induced analgesia.

The blockade of GABA-mediated Cl(-) influx with pentylenetetrazol (PTZ) was used in the present work to induce seizures in animals. The neurotransmission in the postictal period has been the focus of many studies, and there is evidence suggesting antinociceptive mechanisms following tonic-clonic seizures in both animals and men. The aim of this work was to study the involvement of acetylcholine in the antinociception induced by convulsions elicited by peripheral administration of PTZ (64 mg/kg). Analgesia was measured by the tail-flick test in eight albino Wistar rats per group. Convulsions were followed by significant increases in tail-flick latencies (TFLs) at least for 120 min of the postictal period. Peripheral administration of atropine (0.25, 1 and 4 mg/kg) caused a significant dose-dependent decrease in the TFL in seizing animals, as compared to controls. These data were corroborated by peripheral administration of mecamylamine, a nicotinic cholinergic receptor blocker, at the same doses (0.25, 1 and 4 mg/kg) used for the muscarinic cholinergic receptor antagonist. The recruitment of the muscarinic receptor was made 10 min postconvulsions and in subsequent periods of postictal analgesia, whereas the involvement of the nicotinic cholinergic receptor was implicated only after 30 min postseizures. The cholinergic antagonists caused a minimal reduction in body temperature, but did not impair baseline TFL, spontaneous exploration or motor coordination in the rotarod test at the maximal dose of 4 mg/kg. These results indicate that acetylcholine may be involved as a neurotransmitter in postictal analgesia.