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OBJECTIVE: The aim of this investigation was studying the influence of glucose metabolic control on diabetic nephropathy. The authors observed the effect of acarbose, insulin, and both drugs on the metabolic control and development of mesangial enlargement of kidney glomeruli in alloxan-diabetic rats. METHODS: Five groups of Wistar rats were used: normal rats (N), non-treated alloxan-diabetic rats (D), alloxan-diabetic rats treated with acarbose (AD), alloxan-diabetic rats treated with insulin (ID), and alloxan-diabetic rats treated with insulin plus acarbose (IAD). The following parameters were evaluated: body weight; water and food intake; diuresis; blood and urine glucose levels; and the kidney lesions: mesangial enlargement and tubule cell vacuolization. Renal lesions were analysed using a semi-quantitative score 1, 3, 6, 9, and 12 months after diabetes induction. RESULTS: Diabetic rats showed a marked increase of glycemia, urinary glucose levels, diuresis, water and food intake, and weight loss, while the treated diabetic rats showed significant decreased levels of these parameters. The most satisfactory metabolic control was that of diabetic rats treated with acarbose + insulin. There was a significant mesangial enlargement in diabetic rats compared to normal rats from the third up to the 12th month after diabetes induction, with a significant difference between the animals treated with acarbose + insulin and non-treated diabetic rats. A difference between the animals treated with acarbose or insulin alone and non-treated diabetics rats was not seen. CONCLUSIONS: The authors discuss the results stressing the role of diabetic metabolic control in the prevention of diabetic nephropathy.

Background: Contamination of the breathing circuit and medication preparation surface of an anesthesia machine can increase the risk of cross-infection., Objective: To evaluate the contamination of the anesthetic medication preparation surface, respiratory circuits, and devices used in general anesthesia with assisted mechanical ventilation., Design and Setting: Cross-sectional, quantitative study conducted at the surgical center of a philanthropic hospital, of medium complexity located in the municipality of Três Lagoas, in the eastern region of the State of Mato Grosso do Sul., Methods: Eighty-two microbiological samples were collected from the breathing circuits. After repeating the samples in different culture media, 328 analyses were performed., Results: A higher occurrence of E. coli, Enterobacter spp., Pseudomonas spp., Staphylococcus aureus, and Streptococcus pneumoniae (P < 0.001) were observed. Variations were observed depending on the culture medium and sample collection site., Conclusion: The study findings underscore the inadequate disinfection of the inspiratory and expiratory branches, highlighting the importance of stringent cleaning and disinfection of high-touch surfaces.

Guinea pigs are a major source of animal protein for Peruvian Andean families. Despite the economic and cultural relevance of guinea pigs, their genomic characterization has been scarcely addressed. Genotyping-by-sequencing (GBS) has emerged as an affordable alternative to genotyping of livestock and native animals. Here, we report the use of GBS for single nucleotide polymorphism (SNP) discovery of traditionally raised guinea pigs from six regions of the Peruvian Andes and one group of breeding animals. The paired-end (2 × 150 bp) sequencing of 40 guinea pig DNA samples generated a mean of 6.4 million high-quality sequencing reads per sample. We obtained an average sequencing depth of 10× with an 88.5% mapping rate to the Cavia porcellus reference genome. A total of 279 965 SNPs (102 SNPs/Mbp) were identified after variant calling and quality filtering. Based on this SNP set, we assessed the genetic diversity and distance within our selected guinea pig populations. An overall average minor allele frequency of 0.13, an observed heterozygosity of 0.31, an expected heterozygosity of 0.35, and an F-value of 0.1 were obtained, while the SNP-based neighbor-joining tree suggests a closer genetic relationship between individuals from geographically close locations. We showed that GBS is a cost-effective tool for SNP discovery and genetic characterization of Peruvian guinea pig populations. Therefore, it may be considered as a suitable and affordable tool for genomic characterization of poorly studied native animal species., (© 2023 Stichting International Foundation for Animal Genetics.)

Introduction: Some studies have reported the occurrence of microorganisms isolated from water. Considering these microorganisms, fungi are known to occur ubiquitously in the environment, including water, and some are pathogenic and may cause health problems, especially in immunocompromised individuals. The aim of this study was to identify fungi in hospital water samples and to correlate their presence with the concentration of free residual chlorine., Methods: Water samples (100 mL) were collected from taps ( n  = 74) and water purifiers ( n  = 14) in different locations in a university hospital. Samples were filtered through a nitrocellulose membrane and placed on Sabouraud dextrose agar and incubated for 24 hours at 30°C. Fungi were identified according to established methods based on macroscopic and microscopic characteristics (filamentous) and physiological tests (yeasts). Free chlorine residual content was measured at the time of sample collection., Results: Seventy species of fungi were identified in the water samples and about 56% of the water samples contained culturable fungi. Cladosporium oxysporum , Penicillium spinulosum , and Aspergillus fumigatus were the most common filamentous fungi. Aureobasidium pullulans and Candida parapsilosis were the most common yeasts. Chemical analyses revealed that free residual chlorine was present in 81.8% of the samples within recommended concentrations. Among samples from water purifiers, 92.9% showed low levels of free residual chlorine (<0.2 mg/L). There was no significant association between chlorine concentrations (either within or outside the recommended range) and the presence of filamentous fungi and yeasts., Conclusions: This study showed that hospital water can be a reservoir for fungi, some of which are potentially harmful to immunocompromised patients. Free residual chlorine was ineffective in some samples., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this article., (Copyright © 2020 Máira Gazzola Arroyo et al.)

Llamas ( Lama glama ) are invaluable resources of Peru. Despite their importance, their population is decreasing. The Camelid Germplasm Bank-Quimsachata was created as a guardian of this South American camelid (SAC) species and established a bank of llamas from their two types, Ch'aku and Q'ara. However, these populations need to present high genetic diversity to be considered suitable conservation stocks. Thus, in the present study, 13 microsatellites specific for the SAC were used to assess the current genetic variability and differentiation of the llama population from the Bank. The global population showed high genetic diversity with a total of 157 different alleles, with an average of 12.08 alleles per microsatellite, an expected and observed heterozygosity of 0.758 and 0.707, respectively, and an average polymorphic information content (PIC) of 0.723. Although considered as two different breeds and managed separately, the genetic differentiation between Ch'aku and Q'ara was low (F ST = 0.01). Accordingly, the gene flow value was high (Nm = 30.5). Overall, our results indicate the existence of high genetic variation among individuals, and thus, this llama population could be considered a suitable genetic stock for their conservation and for sustainability programs. Additionally, the 13 microsatellites can be used to study other Peruvian llama populations and monitor the genetic variability of llamas from the Camelid Germplasm Bank-Quimsachata.

The importance of dopamine (DA) neurotransmission is emphasized by its direct implication in several neurological and psychiatric disorders. The DA transporter (DAT), target of psychostimulant drugs, is the key protein that regulates spatial and temporal activity of DA in the synaptic cleft via the rapid reuptake of DA into the presynaptic terminal. There is strong evidence suggesting that DAT-interacting proteins may have a role in its function and regulation. Performing a two-hybrid screening, we identified snapin, a SNARE-associated protein implicated in synaptic transmission, as a new binding partner of the carboxyl terminal of DAT. Our data show that snapin is a direct partner and regulator of DAT. First, we determined the domains required for this interaction in both proteins and characterized the DAT-snapin interface by generating a 3D model. Using different approaches, we demonstrated that (i) snapin is expressed in vivo in dopaminergic neurons along with DAT; (ii) both proteins colocalize in cultured cells and brain and, (iii) DAT and snapin are present in the same protein complex. Moreover, by functional studies we showed that snapin produces a significant decrease in DAT uptake activity. Finally, snapin downregulation in mice produces an increase in DAT levels and transport activity, hence increasing DA concentration and locomotor response to amphetamine. In conclusion, snapin/DAT interaction represents a direct link between exocytotic and reuptake mechanisms and is a potential target for DA transmission modulation.

Brain injuries are often associated with the later development of epilepsy. Evidence suggests that morphological and functional changes occur in the remaining neural tissue during a silent (or latent) period in which no seizures are expressed. It is believed that this silent (reorganization) period may provide a therapeutic window for modifying the natural history of disease progression. Here we provide evidence that biperiden, a muscarinic anticholinergic agent, is able to alter disease progression in an animal model of epilepsy. We observed that biperiden was capable of slowing the manifestation of the first spontaneous epileptic seizure and effectively reduced the severity and number of recurrent, spontaneous epileptic seizures during the animals' lifespan. Biomolecular (microdialysis) and electrophysiological (extracellular field recordings) studies determined that biperiden was capable of elevating the threshold of hippocampal excitability, thereby making the hippocampal glutamatergic pathways less responsive to stimuli when high concentrations of potassium were used in vivo or in vitro. Notably, there was no hindrance of long-term memory or learning (a potential problem given the amnestic nature of biperiden). We conclude that biperiden has antiepileptogenic potential and may represent an opportunity for the prevention of post-traumatic epilepsy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Despite the effectiveness of anterior thalamic nucleus (AN) deep brain stimulation (DBS) for the treatment of epilepsy, mechanisms responsible for the antiepileptic effects of this therapy remain elusive. As adenosine modulates neuronal excitability and seizure activity in animal models, we hypothesized that this nucleoside could be one of the substrates involved in the effects of AN DBS. We applied 5 days of stimulation to rats rendered chronically epileptic by pilocarpine injections and recorded epileptiform activity in hippocampal slices. We found that slices from animals given DBS had reduced hippocampal excitability and were less susceptible to develop ictal activity. In live animals, AN DBS significantly increased adenosine levels in the hippocampus as measured by microdialysis. The reduced excitability of DBS in vitro was completely abolished in animals pre-treated with A1 receptor antagonists and was strongly potentiated by A1 receptor agonists. We conclude that some of the antiepileptic effects of DBS may be mediated by adenosine.

Rats with a neonatal ventral hippocampal lesion (NVHL) have been used to model certain features of schizophrenia because they display dopaminergic activity and behavioral alterations consistent with a dysfunctional prefrontal cortex after puberty. Microdialysis studies in normal rats demonstrated increased prefrontal dopamine release during the incentive phase of behavior in an experimental situation specifically designed to evidence this behavioral aspect: the so called "sensory-specific satiety" procedure. Our hypothesis is that if dopaminergic activity in the prefrontal cortex of NVHL rats differs from sham lesioned rats, the responsiveness to the aforementioned experimental situation should also be different. Extracellular medial prefrontal dopamine outflow increased in hungry control rats when they had access to food and decreased across satiety. It increased again when a new food was presented, even when the rats were satiated. NVHL rats also had increased dopamine prefrontal outflow in these conditions, but it remained high after the end of the consumption period. The food consumption behavior declined less rapidly and the reinstatement of food consumption, usually produced by new food, did not occur in NVHL rats, provided the lesions were large. These data were discussed in relation to several theoretical backgrounds developed about the incentive aspect of behavior and for understanding the pathophysiology of schizophrenia., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Deep brain stimulation (DBS) is being investigated as a treatment for major depression, but its mechanisms of action are still unknown. We have studied the effects of ventromedial prefrontal cortex (vmPFC) stimulation in a chronic model of depression and assessed the involvement of the serotonergic system and brain derived neurotrophic factor (BDNF) in a DBS response., Methods: Rats were subjected to chronic unpredictable mild stress during 4 weeks. Decline in preference for sucrose solutions over water, an index suggested to reflect anhedonic-like behavior, was monitored on a weekly basis. The outcome of chronic vmPFC stimulation alone (8 hours/day for 2 weeks) or combined with serotonin-depleting lesions was characterized. BDNF levels were measured in the hippocampus., Results: Stress induced a significant decrease in sucrose preference as well as hippocampal BDNF levels as compared with those recorded in control subjects. vmPFC stimulation completely reversed this behavioral deficit and partially increased BDNF levels. In contrast, DBS did not improve stress-induced anhedonic-like behavior in animals bearing serotonin-depleting raphe lesions with associated normal hippocampal BDNF levels., Conclusions: vmPFC stimulation was effective in a chronic model of depression. Our results suggest that the integrity of the serotonergic system is important for the anti-anhedonic-like effects of DBS but question a direct role of hippocampal BDNF., (Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Excitation of the mesocorticolimbic pathway, originating from dopaminergic neurons in the ventral tegmental area (VTA), may be important for the development of exaggerated fear responding. Among the forebrain regions innervated by this pathway, the amygdala is an essential component of the neural circuitry of conditioned fear. The functional role of the dopaminergic pathway connecting the VTA to the basolateral amygdala (BLA) in fear and anxiety has received little attention. In vivo microdialysis was performed to measure dopamine levels in the BLA of Wistar rats that received the dopamine D(2) agonist quinpirole (1 μg/0.2 μl) into the VTA and were subjected to a fear conditioning test using a light as the conditioned stimulus (CS). The effects of intra-BLA injections of the D(1) antagonist SCH 23390 (1 and 2 μg/0.2 μl) and D(2) antagonist sulpiride (1 and 2 μg/0.2 μl) on fear-potentiated startle (FPS) to a light-CS were also assessed. Locomotor performance was evaluated by use of open-field and rotarod tests. Freezing and increased dopamine levels in the BLA in response to the CS were both inhibited by intra-VTA quinpirole. Whereas intra-BLA SCH 23390 did not affect FPS, intra-BLA sulpiride (2 μg) inhibited FPS. Sulpiride's ability to decrease FPS cannot be attributed to nonspecific effects because this drug did not affect motor performance. These findings indicate that the dopamine D(2) receptor pathway connecting the ventral tegmental area and the basolateral amygdala modulates fear and anxiety and may be a novel pharmacological target for the treatment of anxiety., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Rats with a bilateral neonatal ventral hippocampus lesion (NVHL) are used as models of neurobiological aspects of schizophrenia. In view of their decreased number of GABAergic interneurons, we hypothesized that they would show increased reactivity to acoustic stimuli. We systematically characterized the acoustic reactivity of NVHL rats and sham operated controls. They were behaviourally observed during a loud white noise. A first cohort of 7 months' old rats was studied. Then the observations were reproduced in a second cohort of the same age after characterizing the reactivity of the same rats to dopaminergic drugs. A third cohort of rats was studied at 2, 3, 4, 5 and 6 months. In subsets of lesioned and control rats, inferior colliculus auditory evoked potentials were recorded. A significant proportion of rats (50-62%) showed aberrant audiogenic responses with explosive wild running resembling the initial phase of audiogenic seizures. This was not correlated with their well-known enhanced reactivity to dopaminergic drugs. The proportion of rats showing this strong reaction increased with rats' age. After the cessation of the noise, NVHL rats showed a long freezing period that did neither depend on the size of the lesion nor on the rats' age. The initial negative deflection of the auditory evoked potential was enhanced in the inferior colliculus of only NVHL rats that displayed wild running. Complementary anatomical investigations using X-ray scans in the living animal, and alizarin red staining of brain slices, revealed a thin layer of calcium deposit close to the medial geniculate nuclei in post-NVHL rats, raising the possibility that this may contribute to the hyper-reactivity to sounds seen in these animals. The findings of this study provide complementary information with potential relevance for the hyper-reactivity noted in patients with schizophrenia, and therefore a tool to investigate the underlying biology of this endophenotype.

Background: Subcallosal cingulate gyrus (SCG) deep brain stimulation (DBS) is being investigated as a treatment for major depression. We report on the effects of ventromedial prefrontal cortex (vmPFC) DBS in rats, focusing on possible mechanisms involved in an antidepressant-like response in the forced swim test (FST)., Methods: The outcome of vmPFC stimulation alone or combined with different types of lesions, including serotonin (5-HT) or norepineprhine (NE) depletion, was characterized in the FST. We also explored the effects of DBS on novelty-suppressed feeding, learned helplessness, and sucrose consumption in animals predisposed to helplessness., Results: Stimulation at parameters approximating those used in clinical practice induced a significant antidepressant-like response in the FST. Ventromedial PFC lesions or local muscimol injections did not lead to a similar outcome. However, animals treated with vmPFC ibotenic acid lesions still responded to DBS, suggesting that the modulation of fiber near the electrodes could play a role in the antidepressant-like effects of stimulation. Also important was the integrity of the serotonergic system, as the effects of DBS in the FST were completely abolished in animals bearing 5-HT, but not NE, depleting lesions. In addition, vmPFC stimulation induced a sustained increase in hippocampal 5-HT levels. Preliminary work with other models showed that DBS was also able to influence specific aspects of depressive-like states in rodents, including anxiety and anhedonia, but not helplessness., Conclusions: Our study suggests that vmPFC DBS in rats may be useful to investigate mechanisms involved in the antidepressant effects of SCG DBS.

The involvement of dopamine (DA) mechanisms in the nucleus accumbens (NAC) in fear conditioning has been proposed by many studies that have challenged the view that the NAC is solely involved in the modulation of appetitive processes. However, the role of the core and shell subregions of the NAC in aversive conditioning remains unclear. The present study examined DA release in these NAC subregions using microdialysis during the expression of fear memory. Guide cannulae were implanted in rats in the NAC core and shell. Five days later, the animals received 10 footshocks (0.6 mA, 1 s duration) in a distinctive cage A (same context). On the next day, dialysis probes were inserted through the guide cannulae into the NAC core and shell subregions, and the animals were behaviorally tested for fear behavior either in the same context (cage A) or in a novel context (cage B). Dialysates were collected every 5 min for 90 min and analyzed by high-performance liquid chromatography. The rats exhibited a significant fear response in cage A but not in cage B. Moreover, increased DA levels in both NAC subregions were observed 5-25 min after the beginning of the test when the animals were tested in the same context compared with accumbal DA levels from rats tested in the different context. These findings suggest that DA mechanisms in both the NAC core and shell may play an important role in the expression of contextual fear memory.

The neonatal hippocampus lesion thought to model schizophrenia should show the same modifications in behavioural tests as other models, especially pharmacological models, namely decreased latent inhibition, blocking and overshadowing. The present study is set out to evaluate overshadowing in order to complement our previous studies, which had tested latent inhibition. "Overshadowing" refers to the decreased conditioning that occurs when the to-be-conditioned stimulus is combined with another stimulus at the conditioning stage. We used the same two Pavlovian conditioning paradigms as in our previous works, namely conditioned taste aversion (CTA) and conditioned emotional response (CER). A sweet taste overshadowed a salty conditioned stimulus, and a tone overshadowed a flashing light. Totally different stimuli were used to counter possible sensory biases. The protocols were validated with two groups of Sprague Dawley rats. The same two protocols were then applied to a cohort of rats whose ventral hippocampus had been destroyed when they were 7days old. Only rats with extended ventral hippocampus lesions were included. The overall effect of Pavlovian conditioning was attenuated, significantly so in the conditioned emotional response paradigm, but overshadowing appeared not to be modified in either the conditioned emotional response or the conditioned taste aversion paradigm.

Changes in 5-HT1A receptor-mediated neurotransmission at the level of the median raphe nucleus (MRN) are reported to affect the expression of defensive responses that are associated with generalized anxiety disorder (e.g. inhibitory avoidance) but not with panic (e.g. escape). The objective of this study was to further explore the involvement of MRN 5-HT1A receptors in the regulation of generalized anxiety-related behaviours. Results of experiment 1 showed that intra-MRN injection of the 5-HT1A/7 receptor agonist 8-OH-DPAT (0.6 nmol) in male Wistar rats impaired the acquisition of inhibitory avoidance, without interfering with the performance of escape in the elevated T-maze test of anxiety. Pre-treatment with the 5-HT1A receptor antagonist WAY-100635 (0.18 nmol) fully blocked this anxiolytic-like effect. As revealed by experiment 2, intra-MRN injection of 8-OH-DPAT (0.6, 3 or 15 nmol) also caused anxiolytic effect in rats submitted to the light-dark transition test, another animal model that has been associated with generalized anxiety. In the same test, intra-MRN injection of WAY-100635 (0.18, 0.37 or 0.74 nmol) caused the opposite effect. Overall, the current findings support the view that MRN 5-HT neurons, through the regulation of 5-HT1A somatodendritic autoreceptors, are implicated in the regulation of generalized anxiety-associated behaviours.

The amygdala, the dorsal periaqueductal gray (dPAG), and the medial hypothalamus have long been recognized to be a neural system responsible for the generation and elaboration of unconditioned fear in the brain. It is also well known that this neural substrate is under a tonic inhibitory control exerted by GABA mechanisms. However, whereas there is a growing body of evidence to suggest that the amygdala and dPAG are also able to integrate conditioned fear, it is still unclear, however, how the distinct hypothalamic nuclei participate in fear conditioning. In this work we aimed to examine the extent to which the gabaergic mechanisms of this brain region are involved in conditioned fear using the fear-potentiated startle (FPS). Muscimol, a GABA-A receptor agonist, and semicarbazide, an inhibitor of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD), were used as an enhancer and inhibitor of the GABA mechanisms, respectively. Muscimol and semicarbazide were injected into the anterior hypothalamus (AHN), the dorsomedial part of the ventromedial nucleus (VMHDM), the dorsomedial (DMH) or the dorsal premammillary (PMD) nuclei of male Wistar rats before test sessions of the fear conditioning paradigm. The injections into the DMH and PMD did not produce any significant effects on FPS. On the other hand, muscimol injections into the AHN and VMHDM caused significant reduction in FPS. These results indicate that injections of muscimol and semicarbazide into the DMH and PMD fail to change the FPS, whereas the enhancement of the GABA transmission in the AHN and VMHDM produces a reduction of the conditioned fear responses. On the other hand, the inhibition of this transmission led to an increase of this conditioned response in the AHN. Thus, whereas DMH and PMD are known to be part of the caudal-most region of the medial hypothalamic defensive system, which integrates unconditioned fear, systems mediating conditioned fear select the AHN and VMHDM nuclei that belong to the rostral-most portion of the hypothalamic defense area. Thus, distinct subsets of neurons in the hypothalamus could mediate different aspects of the defensive responses.

Sprague Dawley rats were submitted to bilateral ventral hippocampus lesions 7 days after birth. This corresponds to the Lipska and Weinberger's procedure for modeling schizophrenia. The aim of the present work was to test the learning capacity of such rats with an associative Pavlovian and an instrumental learning paradigm, both methods using reward outcome (food, sucrose or polycose). The associative paradigm comprised also a second learning test with reversed learning contingencies. The instrumental conditioning comprised an extinction test under outcome devaluation conditions. Neonatally lesioned rats, once adults (over 60 days of age), showed a conditioning deficit in the associative paradigm but not in the instrumental one. Lesioned rats remained able to adapt as readily as controls to the reversed learning contingency and were as sensitive as controls to the devaluation of outcome. Such observations indicate that the active access (instrumental learning) to a reward could have compensated for the deficit observed under the "passive" stimulus-reward associative learning condition. This feature is compared to the memory management impairments observed in clinical patients.

Sprague-Dawley rats were submitted to bilateral ventral hippocampus lesions 7 days after birth according to the Lipska and Weinberger's procedure for modeling schizophrenia. The aim of the present work was to better characterize their learning capacity. A double latent inhibition study was conducted using respectively conditioned taste aversion and conditioned emotional response. In the background of this evaluation, locomotion under apomorphine and startle reactions, inhibited or not by prepulses, was also evaluated. Our experimental methods were the same as those used in previous studies from the laboratory which were found to be sensitive to pharmacological manipulations and shown by others to be unaffected by lesions of the ventral hippocampus carried out in adult rats. In contrast, neonatally lesioned rats, once adults (over 60 days old), were hyper-responsive to noise--i.e., the startle response to a 105 db(A) noise pulse was enhanced--and hyperactive under apomorphine (0.7 mg/kg). The prepulse inhibition properties of the startle remained unchanged. Lesioned rats showed a deficit but not a suppression of conditioning, similar in both tests, but latent inhibition was preserved. Such observations complement the already known memory deficit produced in this neurodevelopmental model of schizophrenia.

Rationale: It is well known that 5-HT(2) mechanisms modulate the defensive behavior produced by the stimulation of the dorsal periaqueductal gray (dPAG). However, in spite of the notion that past stressful experiences play a role in certain types of anxiety, only studies with the stimulation of the dPAG of rats without previous aversive experience have been conducted so far., Objectives: We investigated the mediation of 5-HT(2) receptors of the dPAG in rats previously submitted to contextual fear conditioning (CFC). Defensive behaviors induced by the activation of the dPAG were assessed by measuring the lowest intensity of electric current applied to this structure (threshold) able to produce freezing and escape responses during the testing sessions of CFC in which animals were placed in a context previously paired to footshocks. The 5-HT(2) function of the dPAG in this condition was evaluated by local injections of alpha-methyl-5-HT (20 nmol/0.2 mul) and ketanserin (5 and 10 nmol/0.2 mul), selective agonist and antagonist of 5-HT(2) receptors, respectively., Results: In accordance with previous studies, alpha-methyl-5-HT increased the aversive thresholds (antiaversive effects) in naive rats, and injection of ketanserin into the dPAG did not produce significant effects. On the other hand, ketanserin decreased in a dose-dependent manner the freezing threshold (proaversive effect) determined by the dPAG electrical stimulation, whereas alpha-methyl-5-HT continued to show antiaversive effects in animals under CFC., Conclusions: The present results suggest that past stressful experience can produce changes in the synaptic function of 5-HT(2) receptors within the dPAG with important impact on the expression of defensive behaviors.

The inferior colliculus (IC) is involved in processing of auditory information, but also integrates acoustic information of aversive nature. In fact, chemical stimulation of the IC with semicarbazide (SMC) - an inhibitor of the GABA synthesizing enzyme glutamic acid decarboxylase - has been found to cause defensive behavior in an open-field test and functions as an unconditioned stimulus in the place conditioned aversion test (PCA). A question has arisen regarding whether the basolateral nucleus of the amygdala (BLA) is involved in the acquisition of the aversive information ascending from the IC and whether dopaminergic and serotoninergic mechanisms of the BLA regulate this process. Recent evidence has shown that inactivation of the BLA with muscimol inhibits the PCA and causes an increase in the aversiveness of the chemical stimulation of the IC. Based on this, we examined the effects of ketanserin and SCH-23390, antagonists of the 5HT(2) and D(1) receptors, respectively, on the conditioned and unconditioned fear elicited by IC stimulation with SMC. The results obtained confirm the crucial role of 5-HT(2)- and D(1)-mechanisms of the BLA on conditioned fear in that ketanserin and SCH-23390 injections into the BLA caused a reduction in the PCA. On the other hand, ketanserin and SCH-23390 injections into the BLA enhanced the aversiveness of the IC injections of SMC. These findings suggest that while 5-HT(2) and DA(1) mechanisms in the BLA appear to facilitate the conditioned fear they inhibit the unconditioned fear triggered by IC activation.

The neural substrate underlying the aversive effects induced by ethanol abstinence is still unclear. One candidate for such effects is the dorsal periaqueductal gray (dPAG), a core structure of the brain aversion system. The main aim of this study is to examine the role of the dPAG as a possible locus of the aversive effects following abrupt alcohol withdrawal. To this end, rats were subjected to an oral ethanol self-administration procedure, in which animals were offered 6-8% (v/v) ethanol solution for a period of 21 days followed by an abrupt discontinuation of the treatment on the two subsequent days. Control animals received control dietary fluid for similar periods of time. The effects of ethanol withdrawal were examined in the elevated plus-maze (EPM) (Exp. I), on the prepulse inhibition of startle to loud sounds (Exp. II) and on the freezing and escape responses induced by electrical stimulation of the dPAG (Exp. III). In Experiment III, rats were implanted with an electrode aimed at the dPAG and the number and duration of ultrasonic vocalizations (USVs) were also recorded in the rats that received dPAG stimulation at freezing and escape thresholds. Data obtained showed that ethanol withdrawal elicited significant "anxiety-like" behaviors, as revealed by the decrease in the number of entries into and time spent onto the open arms of the EPM. Startle reflex and prepulse inhibition remained unchanged in withdrawn animals. In addition, discontinuation from the chronic ethanol regimen caused a reduction in the stimulation thresholds for freezing and escape and in the number and duration of USVs. Together, these effects have been interpreted in the frame of a high fear state elicited by activation of the dPAG. These findings are indicative that ethanol withdrawal sensitizes the substrates of fear at the level of this midbrain structure.

Chemical stimulation of the inferior colliculus (IC) with semicarbazide--an inhibitor of the gamma aminobutyric acid synthesizing enzyme--functions as an unconditioned stimulus in the conditioned place aversion test (CPA), and electrolytic lesions of the basolateral amygdala (BLA) enhance the aversiveness of the IC stimulation. This study examined the effects of inactivation of the BLA with muscimol on the conditioned and unconditioned fear using semicarbazide injections into the IC of rats subjected to conditioned (CPA) or unconditioned (open field) fear tests. In both tests, the rats were injected with semicarbazide or saline into the IC and muscimol or saline into the BLA. Muscimol decreased the CPA and increased the unconditioned fear elicited by IC injections of semicarbazide. These findings indicate that distinct modulatory mechanisms in the BLA are recruited during the conditioned and unconditioned fear triggered by IC activation., (Copyright 2006 APA, all rights reserved.)

Opioid and serotonergic mechanisms of the ventrolateral periaqueductal gray (vlPAG) are recruited by conditioned freezing and antinociception. However, it is unclear whether freezing and antinociception induced by stimulation of the vlPAG are interrelated. To address this issue we looked at the effects of the opioid antagonist naltrexone, the 5-HT2 antagonist ketanserin, and the benzodiazepine agonist midazolam injected into the vlPAG on the freezing and antinociception induced by electrical stimulation of this region. This antinociception was evaluated by the tail-flick and formalin tests. To further characterize the involvement of the vlPAG in unconditioned fear, the effects of intra-vlPAG injections of midazolam on the exploratory behavior were also assessed in independent groups of rats submitted to the elevated plus-maze test (EPM). The data obtained showed that: (i) electrical stimulation of the vlPAG causes freezing blocked by midazolam but not by naltrexone and ketanserin; (ii) antinociception generated at the level of the vlPAG is inhibited by naltrexone, ketanserin, and midazolam; (iii) activation of benzodiazepine-mediated mechanisms in the vlPAG increased the exploratory behavior of rats in the closed arms but not the avoidance behavior of open arms of the EPM. Thus, freezing and antinociception generated in the vlPAG are dissociated pharmacologically. Whereas antinociception is a multimediated process sensitive to naltrexone, ketanserin, and midazolam, the freezing induced by vlPAG stimulation was reversed only by the benzodiazepine compound. As injections of midazolam into the vlPAG do not cause anxiolytic effects in the EPM, the aversive stimuli inherent of this test seem to bypass the vlPAG.

Rationale: Although 5-HT2 receptors seem to play an important role in anxiety, results from numerous studies are still highly variable. Moreover, little is known about the behavioral effects of centrally administered 5-HT2 compounds in animal models of anxiety., Objective: The current study was performed to: (1) further investigate the effects of 5-HT2 receptor activation in rats exposed to the elevated plus-maze (EPM) and the open-field arena, two widely used animal models for studying anxiety and locomotor activity; and (2) evaluate the involvement of the 5-HT2 receptors within the basolateral nucleus of the amygdala (BLA) in the modulation of such effects., Methods: In the first experiment, male Wistar rats were exposed for 5 min to the EPM 27 min following intraperitoneal (i.p.) (1.0 ml/kg) injections of the preferential 5-HT2C receptor agonist 6-chloro-2[1-piperazinyl]pyrazine (MK-212) at doses of 1.0, 2.0, or 4.0 mg/kg. Control animals were injected with saline. The percentage of open-arm entries and the percentage of time spent in these arms were employed as anxiety indexes, whereas the number of closed-arm entries was calculated as indicative of locomotor activity. In the second experiment, rats were exposed for 10 min in an open-field arena to further assess the interference of the same MK-212 doses upon locomotor activity. In Experiment 3, rats were microinjected (0.2 microl) either with the mixed 5-HT 2A/2C receptor antagonist ritanserin (0.5, 1.25, 2.5, and 5.0 microg) or its vehicle into the BLA 12 min following i.p. injections of saline or the intermediate dose of MK-212 (2.0 mg/kg). Fifteen minutes later, each animal was exposed to the EPM as before., Results: Whereas the highest dose of MK-212 (4.0 mg/kg) induced motor-suppressant effects in both EPM and open-field arena, the intermediate dose of the drug (2.0 mg/kg) reduced open-arm exploration without significantly affecting the number of closed-arm entries. This behavioral profile, consistent with selective anxiogenic effect in the EPM, was dose-dependently prevented by ritanserin microinfusion into the BLA. In saline-pretreated animals, however, ritanserin (all doses) was ineffective., Conclusions: MK-212 increases anxiety and decreases locomotor activity. The anxiogenic-like profile of 5-HT2 receptor activation is prevented by the blockade of 5-HT2 receptors within the BLA, which does not have an effect by itself upon basal anxiety levels triggered by the EPM.

Consistent evidence has shown that dopamine release in the prefrontal cortex is increased by electrical stimulation of the inferior colliculus (IC) as unconditioned stimulus. Recent reports have also demonstrated that inactivation of the basolateral nucleus of the amygdala (BLA) with muscimol enhances the behavioural consequences of the aversive stimulation of the IC and reduces the dopamine release in the prefrontal cortex. Moreover, neurotoxic lesions of the BLA enhance whereas those of the central nucleus of the amygdala (CeA) reduce the aversiveness of the electrical stimulation of the IC. Based on these findings the present study examined the effects of the electrical stimulation of the IC on the extracellular levels of serotonin and dopamine in the BLA and CeA. To this end, rats implanted with a stimulation electrode in the IC also bore a microdialysis probe in the BLA or CeA for determination of the release of dopamine and serotonin. IC electrical stimulation at the freezing and escape thresholds increased the levels of serotonin ( approximately 70%) and dopamine ( approximately 60%) in the BLA related to the basal values. Similarly, the metabolites DOPAC and 5-HIAA increased in a parallel fashion in BLA. No significant changes could be detected in these biogenic amines and metabolites in CeA following IC aversive stimulation. These findings point to a differential role of serotonergic and dopaminergic mechanisms of the BLA and CeA in the setting up of adaptive responses to fear states generated at the inferior colliculus level.

We have shown that stimulation of the neural substrates in the inferior colliculus (IC) causes a significant increase in the extracellular levels of dopamine (DA) in frontal cortex (FC). Also, it has been reported that the basolateral complex of the amygdala (BLA) serves as a filter for unconditioned and conditioned aversive information that ascend to higher structures from the brainstem. Linking these two kinds of information, this work examines whether inactivation of BLA interferes with the activation of cortical dopaminergic outputs produced by aversive stimulation of the IC. To this end, rats were implanted with an electrode in the IC for the determination of the threshold of escape responses. Each rat also bore a cannula implanted in the BLA for injections of lidocaine (10 microg/0.5 microL), muscimol (0.5 microg/0.5 microL), or its vehicle and a microdialysis probe in the FC for determination of the amount of DA and serotonin (5-HT). The data obtained show that IC electrical stimulation caused an increase in the DA release while it reduced the 5-HT release in the FC. BLA inactivation with both lidocaine or muscimol enhanced the aversiveness of the electrical stimulation of the IC and attenuated the increase in DA, while the reduction in 5-HT release in the FC remained unaffected. These findings suggest that ascending aversive information from IC on their way up to higher structures in the SNC courses with opposite modulation by DA/5-HT mechanisms in the FC. These processes are regulated by filters located in the BLA. It is proposed that the loss of these BLA regulatory mechanisms prevents the expression of these modulatory mechanisms in the FC that are adaptive responses in order to cope with unconditioned aversive stimulus triggered at the brainstem level., (copyright (c) 2004 Wiley-Liss, Inc.)

The cell bodies of 5-HT containing neurons that innervate the limbic forebrain are mainly found in the dorsal raphe nucleus and in the median raphe nucleus (MRN). To assess the role of the median raphe nucleus in anxiety, rats bearing either electrolytic or 5-HT-selective neurotoxic lesion of the MRN were tested in the elevated T-maze. This apparatus consists of two opposed open arms perpendicular to one enclosed arm. Two tasks are performed in succession by the same rat in one experimental session, namely inhibitory avoidance of the open arm, taken as a measure of conditioned anxiety and one-way escape from the open arm, considered as a measure of unconditioned fear. The test was performed 7 days after the electrolytic lesion (3 mA, 10s) or 14 days after the neurotoxic lesion (5,7-DHT, 8 microg/1 microl). The results showed that while the electrolytic lesion impaired both inhibitory avoidance and one-way escape, the neurotoxic lesion impaired only inhibitory avoidance. Therefore, serotonergic pathways originating in the MRN seem to participate in the modulation of conditioned anxiety but not unconditioned fear. Other neurotransmitter systems that either originate in or pass through the MRN may regulate unconditioned fear.

Electrical stimulation of the dorsal regions of the periaqueductal gray (PAG) leads to defensive reactions characterized as freezing and escape responses. Until recently it was thought that this freezing behavior could be due to the recruitment of neural circuits in the ventrolateral periaqueductal gray (vlPAG), while escape would be mediated by other pathways. Nowadays, this view has been changing mainly because of evidence that freezing and escape behaviors thus elicited are not altered after lesions of the vlPAG. It has been suggested that there are at least two pathways for periaqueductal gray-mediated defensive responses, one involving the hypothalamus and the cuneiform nucleus (CnF) which mediates responses to immediate danger and another one involving the amygdala and vlPAG which mediates cue-elicited responses, either learned or innate. To examine this issue further we measured Fos protein expression in brain areas activated by electrical stimulation of the dorsolateral PAG (dlPAG) at the freezing and escape thresholds. The data obtained showed that freezing-provoking stimulation caused increases in Fos expression in the dorsomedial PAG (dmPAG), while escape-provoking stimulation led to increases at both dmPAG and dlPAG. Surprisingly, neither escape- nor freezing-provoking stimulations altered Fos expression in the central nucleus of amygdala (CeA). Escape-provoking stimulation caused increased Fos expression in the ventromedial hypothalamus (VMH), dorsal premammilary nucleus (PMd) and in the cuneiform nucleus. Significant increases in Fos labeling were found in the dmPAG and PMd following freezing-provoking stimulation. Therefore, the present data support the notion of a neural segregation for defensive behaviors in the dorsal columns of PAG, with increased Fos expression in the dmPAG following freezing, while dlPAG is affected by both freezing and escape responses. dlPAG, CnF, VMH and PMd are part of a brain aversion network activated by fear unconditioned stimuli. The present data also suggests that the defensive responses generated at the dlPAG level do not recruit the neural circuits of the vlPAG and CeA usually activated by conditioned fear stimuli.

We have shown that 5-HT mechanisms of the median raphe nucleus (MRN) are involved in contextual fear-conditioning processes as electrolytic or neurotoxic lesions with N-methyl-D-aspartate (NMDA) or injections of 8-hydroxy-2-(di-n-propilamino)-tetralin (8-OH-DPAT) into this structure inhibit freezing behavior in a contextual fear paradigm. In this work, we extend these studies by analyzing the behavioral responses in a classical fear-conditioning paradigm (light or tone/foot-shock association) in rats with either neurochemical lesion with NMDA or injected with 8-OH-DPAT into the MRN. The animals received NMDA or 8-OH-DPAT or saline microinjections into the MRN and were submitted to conditioning trials in an experimental chamber, where they received 10 foot-shocks (0.6 mA, 1 s, variable interval between 10 and 50 s) paired with tone or light (CS). On the next day, they were tested in a different experimental chamber, with or without CS presentation, where the duration of freezing and the number of rearing episodes were recorded. Light or tone alone caused a significant amount of freezing. NMDA lesions or 8-OH-DPAT injections into the MRN clearly inhibited freezing behavior in rats conditioned to light/foot-shock association, but not in the conditioning sessions with tones. Besides the proposed role in contextual fear conditioning, these results clearly show that MRN is involved in the fear conditioning with light as conditioned stimuli. Distinct neural substrates seem to subserve conditioning fear with acoustic stimuli.

Rationale: The dorsal periaqueductal gray matter (dPAG) has been implicated in the modulation of defensive behavior. Electrical stimulation of this structure can be used as an unconditioned stimulus to produce a conditioned fear reaction expressed by freezing, antinociception, and autonomic responses., Objectives: This study investigated the influence of benzodiazepine, serotonergic, and opioid mechanisms on these conditioned responses., Methods: Animals implanted with an electrode and a guide cannula into the dPAG were submitted to two conditioning sessions. Each session consisted of ten pairings of the light in a distinctive chamber (CS) with the electrical stimulation of this structure at the escape threshold. On the next day, each animal was exposed only to the CS (testing) and the duration of freezing, number of rearing and grooming episodes were recorded for 5 min. Before and after the testing session, animals were submitted to the tail-flick test. Fifteen minutes before the exposure to the CS, animals received injections into the dPAG of midazolam (a positive modulator of benzodiazepine sites), alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT; an agonist of 5-HT(2) receptors), naltrexone (an opioid antagonist), or vehicle., Results: Conditioning with dPAG electrical stimulation caused significant increases in the time of freezing and conditioned antinociception. Injections of midazolam into the dPAG significantly inhibited freezing behavior and antinociception due to conditioning. Injections of alpha-Me-5-HT inhibited the effects of conditioning on freezing without affecting conditioned antinociception. Injections of naltrexone (13 nmol/0.2 micro l) did not change any of the conditioned responses studied., Conclusions: (1) Conditioned freezing and antinociception are modulated by benzodiazepine mechanisms into dPAG. (2) 5-HT(2) receptors seem to regulate conditioned freezing behavior. However, conditioned antinociception was not affected by 13 nmol naltrexone. (3) Opioid mechanisms do not seem to be involved in the conditioned responses using electrical stimulation of the dPAG as unconditioned stimulus. Further studies with other opioid and 5-HT(2) receptor antagonists are still needed to confirm the conclusions drawn from the present work.

Regulatory mechanisms in the basolateral nucleus of the amygdala (BLA) serves as a filter for unconditioned and conditioned aversive information that ascend to higher structures from the brainstem whereas the central nucleus (CeA) is the main output for the resultant defense reaction. We have shown that neural substrates in the inferior colliculus are activated by threatening stimuli of acoustic nature and have important functional links with the amygdala. In this work, we examined the influence of lesions with 5,7-dihydroxytryptamine (5,7-DHT) of these nuclei of amygdala on the aversive responses induced by electrical stimulation of the inferior colliculus. Thus, rats were implanted with an electrode in the CeA of the inferior colliculus for the determination of the thresholds of alertness, freezing and escape responses. Each rat also bore a cannula implanted in the BLA or CeA for injection of 5,7-DHT (8.0 microg/0.8 microl) or its vehicle. The data obtained show that CeA lesions increase the thresholds of aversive responses whereas BLA lesions decrease the thresholds of these responses. From this evidence it is suggested that defensive behavior induced by activation of the neural substrates of aversion in the inferior colliculus seems to depend on the integrity of the amygdala. BLA regulates the input and CeA functions as the output for these aversive states generated at brainstem level. It is likely that aversive information ascending from the inferior colliculus may receive either inhibitory or excitatory influences of 5-HT mechanisms in the BLA or CeA, respectively., (Copyright 2002 Elsevier Science Inc.)

One widely used animal model of anxiety is the electrical stimulation of a given structure supposed to be involved in the neural circuitry underlying emotional behavior. Indeed, electrical stimulation of midbrain structures with substrates for the processing of fear-like responses, such as the dorsal periaqueductal gray matter (DPAG) or the inferior colliculus (IC), produces behavioral, sensorial and autonomic responses very similar to the defense reactions observed in environmental threatening situations. It has also been proposed that the required level of integration of all these components of the defense reaction needs an integrative process situated at higher brain level, as the prefrontal cortex. As a matter of fact, substantial cortical inputs to the midbrain tectum have already been found. In view of this evidence, it seems important to know whether animals stimulated in the midbrain tectum would present neurochemical changes in the prefrontal cortex. To this end, we examined the temporal course of the effects of the electrical stimulation of the DPAG and IC on the dopamine (DA) release in the prefrontal cortex. Electrical stimulation of these structures was performed at the alertness (control) and escape thresholds. Electrical stimulation of the inferior colliculus at the escape threshold produced a long-lasting increase in the levels of corticofrontal dopamine in relation to these measurements in the control group. No significant changes in extracellular DA release in this cortical area could be observed following DPAG electrical stimulation. These findings bring evidence for the involvement of dopamine of the frontal cortex in the setting up of adaptive responses to stressful situations generated at the inferior colliculus level.