A conserved motif in the immune-subdominant RAP-1 related antigen of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle.

Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis.

Cannabis vapor self-administration elicits sex- and dose-specific alterations in stress reactivity in rats.

RATIONALE: Cannabis users frequently report stress relief as their primary reason for use. Recent studies indicate that human cannabis users exhibit blunted stress reactivity; however, it is unknown whether this is a cause or a consequence of chronic cannabis use. OBJECTIVES: To determine whether chronic cannabis vapor self-administration elicits sex- and/or dose-dependent alterations in stress reactivity and basal corticosterone (CORT) concentrations, or whether pre-vapor exposure stress reactivity predicts rates of cannabis vapor self-administration. METHODS: Male and female rats were subjected to 30 min acute restraint stress to assess stress reactivity prior to vapor self-administration. Rats were then trained to self-administer cannabis extract vapor containing 69.9% Δ9-tetrahydrocannabinol (THC) at one of four extract concentrations (0, 75, 150, or 300 mg/ml) daily for 30 days. Half of the rats were then subjected to a second restraint stress challenge 24 h after the final self-administration session, while the other half served as no-stress controls. Plasma CORT concentrations were measured prior to stress and immediately post-stress offset. RESULTS: Female rats earned significantly more vapor deliveries than male rats. Pre-vapor stress reactivity was not a predictor of self-administration rates in either sex. Basal CORT concentrations were increased following vapor self-administration relative to pre-vapor assessment, irrespective of treatment condition. Importantly, cannabis self-administration dose-dependently reduced stress reactivity in female, but not male, rats. CONCLUSIONS: These data indicate that chronic cannabis use can significantly dampen stress reactivity in female rats and further support the use of the cannabis vapor self-administration model in rats of both sexes.

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats.

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli. The most-widely utilized procedures for studying emotional memories entail fear conditioning in rats. In these tasks, the unconditioned stimulus (US) is a footshock presented once or several times across single or several sessions, and the conditioned response (CR) is freezing. In a version of these procedures, called cued fear conditioning, a tone (conditioned stimulus, CS) is paired with footshocks (US) during the training phase. During the first test, animals are exposed to the same context in which training took place, and freezing responses are tested in the absence of footshocks and tones (i.e., a context test). During the second test, freezing is measured when the context is changed (e.g., by manipulating the smell and walls of the experimental chamber) and the tone is presented in the absence of footshocks (i.e., a cue test). Most cued fear conditioning procedures entail few tone-shock pairings (e.g., 1-3 trials in a single session). There is a growing interest in less common versions involving an extensive number of pairings (i.e., overtraining) related to the long-lasting effect called fear incubation (i.e., fear responses increase over time without further exposure to aversive events or conditioned stimuli). Extended fear-conditioning tasks have been key to the understanding of fear incubation's behavioral and neurobiological aspects, including its relationship with other psychological phenomena (e.g., post-traumatic stress disorder). Here, we describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

Running wheel training does not change neurogenesis levels or alter working memory tasks in adult rats.

BACKGROUND: Exercise can change cellular structure and connectivity (neurogenesis or synaptogenesis), causing alterations in both behavior and working memory. The aim of this study was to evaluate the effect of exercise on working memory and hippocampal neurogenesis in adult male Wistar rats using a T-maze test. METHODS: An experimental design with two groups was developed: the experimental group (n = 12) was subject to a forced exercise program for five days, whereas the control group (n = 9) stayed in the home cage. Six to eight weeks after training, the rats' working memory was evaluated in a T-maze test and four choice days were analyzed, taking into account alternation as a working memory indicator. Hippocampal neurogenesis was evaluated by means of immunohistochemistry of BrdU positive cells. RESULTS: No differences between groups were found in the behavioral variables (alternation, preference index, time of response, time of trial or feeding), or in the levels of BrdU positive cells. DISCUSSION: Results suggest that although exercise may have effects on brain structure, a construct such as working memory may require more complex changes in networks or connections to demonstrate a change at behavioral level.

Effect of Overcrowding on Hair Corticosterone Concentrations in Juvenile Male Wistar Rats.

In many species, chronic stress due to overcrowding during the juvenile period triggers several metabolic and behavioral pathologies in adulthood. The aim of this study was to determine whether a chronic stress condition (overcrowding) induces changes in plasma and hair corticosterone concentrations, overall growth, and organ weights in young Wistar rats. The experimental subjects were divided into 2 groups (control and overcrowded); the overcrowded subjects were exposed to overcrowding during days 38 through 65 after birth. Plasma and hair corticosterone concentrations were higher in overcrowded rats compared with control subjects. In addition, overcrowding reduced body and organ weight gains. These results demonstrate that measuring the concentration of corticosterone in hair samples is an effective, noninvasive method for monitoring chronic stress in rats.

State-dependent auditory evoked hemodynamic responses recorded optically with indwelling photodiodes.

Implantable optical technologies provide measurements of cerebral hemodynamic activity from freely behaving animals without movement constraint or anesthesia. In order to study state-dependent neural evoked responses and the consequential hemodynamic response, we simultaneously measured EEG and scattered light changes in chronically implanted rats. Recordings took place under freely behaving conditions, allowing us to compare the evoked responses across wake, sleep, and anesthetized states. The largest evoked electrical and optical responses occurred during quiet sleep compared to wake and REM sleep, while isoflurane anesthesia showed a large, late burst of electrical activity synchronized to the stimulus but an earlier optical response.

Conditioned lick behavior and evoked responses using whisker twitches in head restrained rats.

To examine whisker barrel evoked response potentials in chronically implanted rats during behavioral learning with very fast response times, rats must be calm while immobilized with their head restrained. We quantified their behaviors during training with an ethogram and measured each individual animals' progress over the training period. Once calm under restraint, rats were conditioned to differentiate between a reward and control whisker twitch, then provide a lick response when presented with the correct stimulus, rewarded by a drop of water. Rats produced the correct licking response (after reward whisker twitch), and learned not to lick after a control whisker was twitched. By implementing a high-density 64-channel electrocorticogram (ECoG) electrode array, we mapped the barrel field of the somatosensory cortex with high spatial and temporal resolution during conditioned lick behaviors. In agreement with previous reports, we observe a larger evoked response after training, probably related to mechanisms of cortical plasticity.

Discrimination of auditory stimuli during isoflurane anesthesia.

Deep isoflurane anesthesia initiates a burst suppression pattern in which high-amplitude bursts are preceded by periods of nearly silent electroencephalogram. The burst suppression ratio (BSR) is the percentage of suppression (silent electroencephalogram) during the burst suppression pattern and is one parameter used to assess anesthesia depth. We investigated cortical burst activity in rats in response to different auditory stimuli presented during the burst suppression state. We noted a rapid appearance of bursts and a significant decrease in the BSR during stimulation. The BSR changes were distinctive for the different stimuli applied, and the BSR decreased significantly more when stimulated with a voice familiar to the rat as compared with an unfamiliar voice. These results show that the cortex can show differential sensory responses during deep isoflurane anesthesia.

In vitro and in vivo noise analysis for optical neural recording.

Laser diodes (LD) are commonly used for optical neural recordings in chronically recorded animals and humans, primarily due to their brightness and small size. However, noise introduced by LDs may counteract the benefits of brightness when compared to low-noise light-emitting diodes (LEDs). To understand noise sources in optical recordings, we systematically compared instrument and physiological noise profiles in two recording paradigms. A better understanding of noise sources can help improve optical recordings and make them more practical with fewer averages. We stimulated lobster nerves and a rat cortex, then compared the root mean square (RMS) noise and signal-to-noise ratios (SNRs) of data obtained with LED, superluminescent diode (SLD), and LD illumination for different numbers of averages. The LED data exhibited significantly higher SNRs in fewer averages than LD data in all recordings. In the absence of tissue, LED noise increased linearly with intensity, while LD noise increased sharply in the transition to lasing and settled to noise levels significantly higher than the LED's, suggesting that speckle noise contributed to the LD's higher noise and lower SNRs. Our data recommend low coherence and portable light sources for in vivo chronic neural recording applications.

Evoked response potential markers for anesthetic and behavioral states.

The rodent whisker sensory system is a commonly used model of cortical processing; however, anesthetics cause profound differences in the shape and timing of evoked responses. Evoked response studies, especially those that use spatial mapping techniques, such as fMRI or optical imaging, will thus show significantly different results depending on the anesthesia used. To describe the effect of behavioral states and commonly used anesthetics, we characterized the early surface-evoked response potentials (ERPs) components (first ERP peak: gamma band 25-45 Hz; fast oscillation: 200-400 Hz; and very fast oscillation: 400-600 Hz) using a 25-channel electrode array on the somatosensory cortex during whisker stimulation. We found significant differences in the ERP shape when ketamine/xylazine, urethane, propofol, isoflurane, and pentobarbital sodium were administered and during sleep and wake states. The highest ERP amplitudes were observed under propofol anesthesia and during quiet sleep. Under isoflurane, the ERP was nearly absent, except for a very late component, which was concombinant with burst synchronization. The slowest responses were seen under urethane and propofol anesthesia. Spatial mapping experiments that use electrical, NMR, or optical techniques must consider the anesthetic dependency of these signals, especially when stimulation protocols or electrical and metabolic responses are compared.

Local functional state differences between rat cortical columns.

Surface evoked potentials (SEPs) during auditory clicks and whisker twitches are usually larger during quiet sleep (QS) over waking and REM sleep. However, SEP amplitudes from single trials fluctuate periodically between high and low values regardless of sleep-wake cycle. To test the hypothesis that state-independent fluctuations represent local functional sleep-like states of individual cortical columns, we examined single trial SEP amplitudes from multiple cortical locations across sleep-wake cycles. Bilateral stimuli produced SEP amplitude fluctuations in each hemisphere that usually covaried (r = 0.4), but with frequent hemispheric differences. Two neighboring whiskers, twitched simultaneously on the same side, produced highly correlated SEPs in neighboring cortical columns (r = 0.9) with frequent divergences. We found 50% more disparity during QS over waking, indicating that the differences did not result from recording noise or stimulus inconsistency. Local SEP fluctuations also followed local differences in the delta wave signal during QS (r = 0.4), suggesting that similar mechanisms may modulate the SEP. The duration of the localized sleep-like (high SEP amplitude) state was dependent on the duration of prior wake-like (low SEP amplitude) state (r = 0.5), suggesting a use dependence of prior functional state period. Since SEP indicators fluctuated independently from whole animal sleep state, and were frequently different between hemispheres and nearby cortical columns, these data support the theory that sleep-like functional states may be localized to brain regions at least as small as cortical columns.