Select pyrethroid binary mixtures (deltamethrin plus S-bioallethrin, β-cyfluthrin, cypermethrin, and fenpropathrin) elicit a more-than-additive response on L-glutamate release from rat brain synaptosomes that is independent of calcium influx. Using a variety of chloride channel antagonists, anthracene-9-carboxylic acid (9-AC), rChlorotoxin (ClTx), 4,4’-dintitrostilbene-2,2’-disulfonic acid (DNDS), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), and picrotoxinin (PTX), we have identified two mechanisms by which pyrethroids may enhance L-glutamate release. The results from this study indicate that only ClTx and NPPB, at their EC50s (0.1 μM and 70 μM, respectively), significantly increase L-glutamate release when in the presence of our most potent pyrethroid, deltamethrin, at its EC50 (2 x 10-12 M). When these two antagonists were used in the presence of deltamethrin plus cypermethrin and deltamethrin plus fenpropathrin, a more-than-additive response was elicited at lower concentrations of the binary mixtures. Likewise, NPPB in the presence of the additive binary mixture, deltamethrin plus tefluthrin, first elicited a more-than-additive response at the 1:10 mixture. Since both ClTx and NPPB are inhibitors of voltage-gated chloride channels (ClC-2) and calcium-activated chloride channels, our findings suggest that these channels are potential target sites for certain pyrethroids and likely are important in pyrethroid neurotoxicity.

Pain is a complex neuro-psychosocial experience that is internal and private making it difficult to assess in both humans and animals. In research approximately 95% of animal models use rodents, with rats being among the most common for pain studies [3]. However, traditional assessments of the pain response struggle to demonstrate that the behaviors are a direct measurement of pain. The rat grimace scale (RGS) was developed based on facial action coding systems (FACS) which have known utility in non-verbal humans [6, 9]. The RGS measures facial action units of orbital tightening, ear changes, nose flattening, and whisker changes in an attempt to quantify the pain behaviors of the rat. These action units are measured on frontal images of rats with their face in clear view on a scale of 0-2, then summed together. The total score is then averaged to find a final value for RGS between 0-2. Currently, the software program Rodent Face Finder® can extract frontal face images. However, the RGS scores are still manually recorded which is a labor-intensive process, requiring hours of training. Furthermore, the scoring can be subjective, with differences existing between researchers and lab groups. The primary aim of this study is to develop an automated system that can detect action unit regions and generate a RGS score for each image. To accomplish this objective, a YOLOv5 object detector and Vision Transformers (ViT) for classification were trained on a dataset of frontal-facing images extracted using Rodent Face Finder®. Subsequently, the model was then validated using a RGS test for blast traumatic brain injury (bTBI). The validation dataset consisted of 40 control images of uninjured rats, 40 images from the bTBI study on the day of injury, and 40 images 1-month post-injury. All 120 images in the validation set were then manually graded for RGS and tested using the automated RGS system. The results indicated that the automated RGS system accurately and efficiently graded the images with minimal variation in results compared to human graders in just 1/14th of the time. This system provides a fast and reliable method to extract meaningful information of rats' internal pain state. Furthermore, the study presents an avenue for future research into real-time pain monitoring. / Master of Science / Pain is a difficult experience to measure, both in humans and animals. It can be a subjective experience that is largely based on individual perception and interpretation. Furthermore, in animals, pain is even more challenging to assess because they cannot communicate their experience through language. Nonetheless, animal research plays an important role in understanding and treating the underlying mechanisms of pain. In animal research, rats are commonly used to study pain. However, traditional methods of assessing pain behaviors are not meant to observe the pain experience, but instead analyze a response to an external stimulus. The rat grimace scale (RGS) was developed as a direct measurement of the pain experience by analyzing the facial features. Currently, RGS scores are manually recorded by trained researchers which is time-consuming and can be subjective. This study aimed to develop an automated system to identify pain related facial expressions and generate a RGS score for frontal-images of rats. The system was trained using a dataset of frontal-facing rat images with varying levels of RGS scores and validated using images of rats from a traumatic brain injury study. The results showed that the automated RGS system accurately identified RGS pain level differences between recently injured rats, uninjured rats, and rats which were allowed to recover for 1-month. Furthermore, the system provided a fast and reliable method for measuring rat pain behavior when compared to manual grading. With this system, researchers will be able to efficiently perform RGS test. Additionally, this study presents an opportunity for future automation of other grimace scales as well as research into real-time pain monitoring.

This study investigated the β-subunit of the GABAa receptor and determine if there are changes in the primary sequence of the extracellular N-Terminal domain, which likely regulates GABA binding.

Naked mole-rats live in large colonies and exhibit a strict reproductive hierarchy. Each colony has 1 breeding female and 1-3 breeding males; all other individuals are non-reproductive subordinates. Subordinates show a remarkable lack of sex differences in behavior and anatomy, but can become reproductive if removed from the colony. The striated perineal muscles and their innervating motoneurons, which are sexually dimorphic in all other mammals examined, are not dimorphic in subordinate naked mole-rats. Here I asked whether sexual differentiation of this neuromuscular system occurs when subordinates become breeders. Sex differences in perineal motoneurons were not observed, regardless of social status. To my surprise counts of motoneurons in Onuf’s nucleus were increased in breeders of both sexes. This was accompanied by a reciprocal decrease in cells in Onuf’s nucleus that were characterized by small soma size. The neuronal changes correlate with increased perineal muscle volumes in breeders. Although not exhibiting typical motoneuron morphology, some small cells fit a neurochemical or functional definition of a motoneuron. I propose that small cells are recruited to the pool of large Onuf’s nucleus motoneurons when subordinate naked mole-rats become breeders. I then looked at naked mole-rats of varying status (subordinates, paired animals that have never reproduced, intact breeders, and gonadectomized breeders) to determine which cues elicit changes in perineal muscles and small cells in Onuf’s nucleus. I found that pairing is sufficient to cause decreases in the population of small cells in Onuf’s nucleus, while production of litters is necessary for increasing in perineal muscle size. The gonads were not necessary to maintain changes in small cells or perineal muscles. I hypothesized that the lack of sex differences in naked mole-rats might be related to their unusual social structure. To test this, I compared the genitalia and perineal muscles in three African mole-rat species: the naked mole-rat, the solitary silvery mole-rat, and the Damaraland mole-rat, a species considered to be eusocial, but with less reproductive skew than naked mole-rats. My findings support a relationship between social structure, mating system, and sexual differentiation.

<P> Mitochondria contain DNA which is distinct from nuclear DNA. The capacity of isolated rat liver mitochondria to synthesize RNA and the types of RNA synthesized were examined in order to determine the genetic function of mitochondrial DNA. </p> <p> It was demonstrated that isolated rat liver mitochondria synthesize RNA, incorporating [3H]UTP, [3H] ATP,[3H]CTP and [3H]GTP in a DNA-dependent reaction. In addition, the DNA-independent incorporation of [3H]CTP and [3H]ATP suggested metabolic turnover of the CCA end of mitochondrial tRNA. </p> <P> Analysis of the , newly-synthesized RNA by sucrose density gradient centrifugation and agarose-polyacrylamide gel electrophoresis demonstrated that mitochondrial ribosomal RNA was synthesized in a DNA-dependent process. It is concluded that one of the genetic functions of mitochondrial DNA is to code for mitochondrial ribosomal RNA. </p> / Thesis / Doctor of Philosophy (PhD)

<p> Body temperature regulation at high ambient temperatures was compared in pregnant and non-pregnant female albino rats. At an ambient temperature of 40°C, pregnant rats maintained lower body temperatures than non-pregnant rats without added expenditure of moisture for evaporative cooling. This was surprising because of the increased heat load on the pregnant animal resulting from a large weight gain and increased food intake. The maintenance of lower body temperatures in the heat by pregnant rats was possible because (1) pregnant rats produce less heat; i.e. consume less oxygen during exposure to 40°C than do non-pregnant rats and thus need to dissipate less heat; and (2) pregnant rats have a lower body temperature threshold for increased submaxillary salivary gland output in the heat, which makes more water for evaporative cooling available to them at lower body temperatures.</p> <p> The maintenance of lower body temperatures in the heat by pregnant rats suggests that pregnant rats have a need or a preference for lower body temperatures in the heat. The change in body temperature regulation during pregnancy is likely related to physiological and anatomical changes in the body which accompany pregnancy. Alterations in body temperature regulation to meet the changed physiological state of pregnancy provide another example of the body's remarkable ability to maintain homeostasis.</p> / Thesis / Doctor of Philosophy (PhD)

<p> Neonatal rats exposed to heat stress were studied to determine the age at which the saliva-spreading response appears, and to elucidate any other heat defenses that might exist before the response develops.</p> <p> Saliva-spreading appeared on the 17th day of age, the age at which hypothalamic maturity is attained. This finding thus agrees with previous hypotheses that the hypothalamus is involved in the regulation of body temperature.</p> <p> The tolerance of rats to an ambient temperature of 40 C dropped from 16 - 26 hours to 2 - 4 hours in the first 10 days of life. Three factors accounted for this change: decreased body water, increased rates of water loss, and increased metabolic rate.</p> / Thesis / Master of Arts (MA)

Indiana University-Purdue University Indianapolis (IUPUI) / Problematic alcohol consumption develops concurrently with deficits in decision-making. These deficits may be due to alterations in dorsal medial prefrontal cortex (dmPFC) neural activity, as it is essential for the evaluation and implementation of behavioral strategies. In this study, we hypothesized that differences in cognitive control would be evident between Wistars and alcohol-preferring P rats. Cognitive control can be split into proactive and reactive components. Proactive control maintains goal-directed behavior independent of a stimulus whereas reactive control elicits goal-directed behavior at the time of a stimulus. Specifically, it was hypothesized that Wistars would show proactive control over alcohol-seeking whereas P rats would show reactive control over alcohol-seeking. Proactive control in our rodent model is defined as responding to distal task cues whereas reactive control is responding to proximal cues. This was tested in rodents performing a 2-way Cued Access Protocol (2CAP) that facilitates measurements of alcohol seeking and drinking. Congruent sessions were the typical, default 2CAP sessions that consisted of the CS+ being on the same side as alcohol access. These were compared with incongruent sessions where alcohol access was opposite of the CS+. Wistars exhibited an increase in incorrect approaches during the incongruent sessions, which was not detectable in P rats. A trial-by-trial analysis indicated that the increases in incorrect responses was explained by Wistars utilizing the previously learned task-rule, whereas the P rats did not. This motivated the subsequent hypothesis that neural activity patterns corresponding to proactive control would be observable in Wistars but not P rats. Principal Component Analysis indicated that neural ensembles in the dmPFC of Wistars exhibited decreased activity to the cue light in incongruent sessions whereas P rat ensembles displayed increased activity at timepoints associated with the onset and end of alcohol access. Overall, it was observed that P rats showed the most differences in neural activity at times relevant for alcohol delivery; specifically, when the sipper came into the apparatus and left. Conversely, Wistars showed differences prior to approach as evidenced by both differences in cue-related activity as well as differences in spatial-strategies. Together, these results support our hypothesis that Wistars are more likely to engage proactive cognitive control strategies whereas P rats are more likely to engage reactive cognitive control strategies. Although P rats were bred to prefer alcohol, differences in cognitive control phenotypes may have concomitantly occurred that are of clinical relevance.

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