Oral administration of encapsulated catechin in chitosan-alginate nanoparticles improves cognitive function and neurodegeneration in an aluminum chloride-induced rat model of Alzheimer's disease.

The present study aimed to investigate the effect of catechin-loaded Chitosan-Alginate nanoparticles (NPs) on cognitive function in an aluminum chloride (AlCl3)-induced rat model of Alzheimer's disease (AD). The Catechin-loaded Chitosan-Alginate nanocarriers were synthesized through ionotropic gelation (IG) method. Physio-chemical characterization was conducted with the Zetasizer Nano system, the scanning electron microscope, and the Fourier transform infrared spectroscopy. The experiments were performed over 21 days on six groups of male Wistar rats. The control group, AlCl3 treated group, Catechin group, nanocarrier group, treatment group 1 (AlCl3 + Catechin), and treatment group 2 (AlCl3 + nanocarrier). A behavioral study was done by the Morris water maze (MWM) test. In addition, the level of oxidative indices and acetylcholine esterase (AChE) activity was determined by standard procedures at the end of the study. AlCl3 induced a significant increase in AChE activity, along with a significant decrease in the level of Catalase (CAT) and total antioxidant capacity (TAC) in the hippocampus. Moreover, the significant effect of AlCl3 was observed on the behavioral parameters of the MWM test. Both forms of Catechin markedly improved AChE activity, oxidative biomarkers, spatial memory, and learning. The present study indicated that the administration of Catechin-loaded Chitosan-Alginate NPs is a beneficial therapeutic option against behavioral and chemical alteration of AD in male Wistar rats.

Role of Necroptosis, a Regulated Cell Death, in Seizure and Epilepsy.

Epilepsy is a chronic neurological disease that is characterized by spontaneous and recurrent seizures. Regulated cell death is a controlled process and has been shown to be involved in neurodegenerative diseases. Necroptosis is a type of regulated cell death, and its association with epilepsy has been documented. Necroptosis signaling can be divided into two pathways: canonical and non-canonical pathways. Inhibition of caspase-8, dimerization of receptor-interacting protein kinase 1 (RIP1) and RIP3, activation of mixed-lineage kinase domain-like protein (MLKL), movement of MLKL to the plasma membrane, and cell rupture occurred in these pathways. Through literature review, it has been revealed that there is a relationship between seizure, neuroinflammation, and oxidative stress. The seizure activity triggers the activation of various pathways within the central nervous system, including TNF-α/matrix metalloproteases, Neogenin and Calpain/ Jun N-terminal Kinase 1, which result in distinct responses in the brain. These responses involve the activation of neurons and astrocytes, consequently leading to an increase in the expression levels of proteins and genes such as RIP1, RIP3, and MLKL in a time-dependent manner in regions such as the hippocampus (CA1, CA3, dentate gyrus, and hilus), piriform cortex, and amygdala. Furthermore, the imbalance in calcium ions, depletion of adenosine triphosphate, and elevation of extracellular glutamate and potassium within these pathways lead to the progression of necroptosis, a reduction in seizure threshold, and increased susceptibility to epilepsy. Therefore, it is plausible that therapeutic targeting of these pathways could potentially provide a promising approach for managing seizures and epilepsy.

Role of Pyroptosis, a Pro-inflammatory Programmed Cell Death, in Epilepsy.

Epilepsy is one of the most common serious brain diseases worldwide. Programmed cell death (PCD), a cellular self-destruction phenomenon, has been greatly documented in neurodegenerative diseases. Pyroptosis is a well-known pro-inflammatory PCD, and its involvement in epilepsy has been reported in animal models of epilepsy and also epileptic patients. Canonical (caspase-1-dependent) and non-canonical (caspase-1-independent) pathways are two main mechanisms implicated in pyroptotic cell death. Mouse caspase-11 or human analogues caspase-4/5 induce the non-canonical pathway. In both pathways, membrane gasdermin (GSDMD) pores contribute to pro-inflammatory cytokine release and lead to membrane destabilization and cell lysis. IL-1ß and IL-18 are pro-inflammatory cytokines that are released following pyroptotic PCD. Brain inflammation increases excitability in the nervous system, promotes seizure activity, and is probably associated with the molecular and synaptic changes involved in epileptogenesis. Pro-inflammatory cytokines affect the glutamate and GABA neurotransmitter release as well as their receptors, thereby resulting in seizure activity. This review is intended to provide an overview of the current published works on pyroptotic cell death in epilepsy. The mechanisms by which pro-inflammatory cytokines, including IL-1ß and IL-18 can promote epileptic discharges were also collected. According to this survey, since the involvement of pyroptosis in the development of epilepsy has been established, pyroptosis-targeted therapies may represent a novel anti-epileptogenic strategy.

Involvement of orexin-2 receptors in the CA1 region of the hippocampus in the extinction and reinstatement of methamphetamine-induced conditioned place preference in the rats.

The present study, regarding the orexin receptors having a pivotal role in reward-related psychostimulant use disorder (PUD), aimed to investigate the role of orexin-2 (OX2) receptors in the CA1 region of the hippocampus (HPC) in the extinction and reinstatement of methamphetamine (METH)-induced conditioned place preference (CPP). In the first set of investigations, to determine the role of OX2 receptors in the extinction of METH-induced CPP, rats were daily given (during the extinction) bilaterally intra-CA1 region different doses of TCS OX2 29 (1, 3, 10, and 30 nmol/0.5 µl 12% DMSO) as the selective OX2 receptor antagonist. Then, to demonstrate the role of OX2 receptors in the reinstatement of METH-induced CPP after the extinction was established, each rat bilaterally received TCS OX2 29 at the same doses in the CA1 region before injection of the sub-threshold (priming) dose of METH (0.25 mg/kg, sc) on the reinstatement day. The data revealed that the administration of TCS OX2 29 in the CA1 region reduces the mean extinction latency and suppresses the reinstatement of METH-seeking behavior in extinguished rats. Additionally, the potency of TCS OX2 29 to inhibit the reinstatement phase was higher compared to the potency of this drug to modulate the extinction phase of METH-induced CPP. Accordingly, it could be concluded that the blockade of the OX2 receptors in this area might be an essential application and potential therapeutics in treating METH use disorder.

The Role of Orexin-1 Receptors Within the Hippocampal CA1 Area in the Extinction and Reinstatement of Methamphetamine-Seeking Behaviors.

Psychostimulant addiction is a chronic brain disorder with high relapse rates, requiring new therapeutic strategies. The orexin system is highly implicated in processing reward and addiction through connections with critical areas such as the hippocampus. This study investigated the role of orexin-1 receptors (OX1R) within the CA1 subregion of the hippocampus in the extinction and reinstatement of the methamphetamine-induced conditioned place preference. After cannulae implantation, recovery, and establishing the methamphetamine place preference, 98 male Wistar rats received different doses of bilateral intra-CA1 selective OX1R antagonist, SB334867 (1, 3, 10, and 30 nmol/0.5 µl DMSO per side) during the 10-day extinction period (daily) or after extinction phase, just on the reinstatement day (single dose) in separate experimental and control groups. The findings indicated that bilateral microinjection of SB334867 into the CA1 area during the extinction period could significantly reduce the extinction latency and maintenance of rewarding aspects of methamphetamine dose-dependently (3, 10, and 30 nmol). In another set of experiments, a single dose of bilateral intra-CA1 SB334867 administration on the reinstatement phase prevented the methamphetamine-induced reinstatement of drug-seeking behaviors at the high doses (10, and 30 nmol). The present study provided more evidence for the implication of hippocampal OX1R in the maintenance of rewarding and reinforcing properties of methamphetamine and its role in the relapse of methamphetamine-seeking behavior. Further investigations on the role of the orexin system, including the orexin-2 receptors in treating addiction, are needed to introduce its antagonists as effective therapeutic options for psychostimulant addiction.

Dementia in diabetes mellitus and atherosclerosis: Two interrelated systemic diseases.

Dementia is a common problem among the elderly and is defined by the reduction in memory and cognition. Dementia is often caused by a neurodegenerative disease such as Alzheimer's disease, but a number of systemic disorders would also lead to this devastating condition. It is necessary to identify these disorders because many of them are treatable. This article provides an overview of the relationship between systemic disorders, including diabetes mellitus and atherosclerosis, with the development of dementia; two common systemic disorders that their association with each other and also with dementia has been established in many previously published articles. Additionally, the mechanisms involved in the pathogenesis of dementia via the quoted disorders were discussed almost in detail.

Thyroid Disorders and Development of Cognitive Impairment: A Review Study.

Dementia is a neurological disorder that is spreading with increasing human lifespan. In this neurological disorder, memory and cognition are declined and eventually impaired. Various factors can be considered as the background of this disorder, one of which is endocrine disorders. Thyroid hormones are involved in various physiological processes in the body; one of the most important of them is neuromodulation. Thyroid disorders, including hyperthyroidism or hypothyroidism, can affect the nervous system and play a role in the development of dementia. Despite decades of investigation, the nature of the association between thyroid disorders and cognition remains a mystery. Given the enhancing global burden of dementia, the principal purpose of this study was to elucidate the association between thyroid disturbances as a potentially modifiable risk factor of cognitive dysfunction. In this review study, we have tried to collect almost all of the reported mechanisms demonstrating the role of hypothyroidism and hyperthyroidism in the pathogenesis of dementia.

Intraventricular insulin adjacent to the arcuate nucleus reduced the formalin-induced pain through dorsal raphe nucleus opioid receptors in the STZ-induced diabetic rats.

Diabetes mellitus is one of the diseases that affect nociception. In type 1 diabetes, the insulin release declines. One of the regions that respond to insulin and have insulin receptors is the hypothalamus, especially the arcuate nucleus. This hypothalamic nucleus has proopiomelanocortin (POMC)-containing neurons that affect the pain endogenous modulatory pathways such as dorsal raphe nucleus (DR) via releasing endorphins. So it was tried to investigate the influence of insulin within the arcuate nucleus with/without DR opioid receptors blockade on the nociception in the formalin test paradigm. In the present study, the role of different doses of insulin (2, 10, and 50 mIU/0.5 µl saline) within the arcuate nucleus was investigated via formalin test in type 1 (STZ-induced) diabetic rats. To perform the formalin test, 50 µl of formalin 2.5% was injected subcutaneously (s.c.) into the right palm. The behavior of the animal after the stimulation of pain receptors by s.c. formalin injection was scored from 0 (no distinguished pain) to 3 (the most nociception and highest pain score). Insulin within the arcuate nucleus diminished the nociception in formalin-induced paw in the STZ-induced diabetic rats. Intra-DR naloxone 0.2 µg/0.5 µl saline prevented this analgesia. A possible suggested mechanism for this observation is that insulin reinforces the POMC and endorphin release from the arcuate nucleus and decreases pain through DR.

The role of dentate gyrus dopaminergic receptors in the lateral hypothalamic-induced antinociception during persistent inflammatory pain in male rats.

The lateral hypothalamus (LH) is one of the key brain areas involved in pain modulation. Also, the dentate gyrus (DG) of the hippocampus expresses various receptors, including dopaminergic receptors. Dopaminergic receptors play a key role in pain transmission and modulation within the brain. The present study aimed to investigate the involvement of DG dopaminergic receptors in the LH-induced antinociception during the presence of inflammatory pain. Male Wistar rats were used in this study. Cannulae were unilaterally implanted in their skull for microinjections into the LH and DG. The LH was chemically stimulated by carbachol injection (250 nM/0.5 µl saline). In separate groups, different doses (0.25, 1, and 4 µg/0.5 µl vehicle) of the D1- and D2-like dopamine receptor antagonists (SCH23390 and Sulpiride, respectively) were microinjected into the DG, 5 min prior to intra-LH injection of carbachol. Five min after the second injection, formalin test as a persistent inflammatory pain model in animals was done in all rats. The results revealed that carbachol could induce antinociception following formalin injection into rat's hind paw. The 4 µg dose of both antagonists significantly reduced the LH stimulation-induced antinociception in both phases of formalin pain responses. Although the 1 µg dose of sulpiride significantly reduced antinociception during both phases, 1 µg SCH23390 could only reduce this antinociception during the late phase. These findings demonstrate the involvement of DG dopaminergic receptors in the LH-induced antinociception. The results also suggest that the effectiveness of DG dopaminergic receptors is more pronounced during the late phase of formalin-induced pain responses.

A literature overview on epilepsy and inflammasome activation.

Epilepsy is one of the most prevalent serious brain disorders worldwide. Accumulating evidence has suggested that inflammation participates in the progression and pathogenesis of epilepsy. During inflammation, a cytosolic multimolecular complex called the "inflammasome" is activated, driving the innate immune response. This inflammatory pathway by sensing various pathogens and molecules from damaged cells and then activation of caspase-1 enzyme initiates inflammatory responses. Activated caspase-1 leads to the proteolytic cleavage of the pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and interleukin-18 (IL-18), and also induction of an inflammatory programmed cell death termed pyroptosis. NLR family pyrin domain-containing 1 (NLRP1) and NLRP3 are the two best-characterized inflammasome members, and both basic and clinical research has reported their activation during epilepsy. This overview is intended to summarize the current literature concerning NLRP1 and NLRP3 inflammasome activation and epilepsy.

Role of D1- and D2-like dopamine receptors within the dentate gyrus in antinociception induced by chemical stimulation of the lateral hypothalamus in an animal model of acute pain.

The dentate gyrus (DG) as the main gateway of the hippocampal formation (HF) plays a crucial role in pain modulation. For this purpose, the HF receives dopaminergic inputs originated from the substantia nigra and the ventral tegmental area. It has previously been shown that the lateral hypothalamus (LH) stimulation produces antinociception via orexinergic projections of the LH to the DG region. So, given the presence of dopamine receptors in the DG and the undeniable role of the dopaminergic system in pain modulation, the current study was conducted to investigate the role of dopamine receptors located within the DG in the LH stimulation-induced pain modulation. Adult male Wistar rats weighing 220-250 g were unilaterally implanted with two separate cannulas into the LH and DG. Intra-DG administration of D1- or D2-like dopamine receptor antagonist (0.125, 0.25, 1, and 4 µg) was performed just 5 min before chemical stimulation of the LH by carbachol (250 nM). Nociceptive assay was done using the tail-flick apparatus immediately after the last microinjection. The results demonstrated that the administration of SCH23390 or Sulpiride into the DG decreased the intra-LH carbachol-induced antinociceptive responses and decreased the tail-flick latency times. The role of D2-like dopamine receptor of the DG was more prominent than that of D1-like dopamine receptor in antinociceptive response produced by the LH stimulation. It seems to be a complex neural circuitry in which the LH produces antinociceptive effects, in part, via dopamine receptors located in the DG region.

Insulin within the Arcuate Nucleus Has Paradoxical Effects on Nociception in Healthy and Diabetic Rats.

INTRODUCTION: Broad neural circuits originate from the hypothalamic arcuate nucleus and project to many parts of the brain which are related to pain perception. Insulin receptors are found in the arcuate nucleus. Since nociception may be affected in type 1 diabetes, the present study aimed to investigate the intra-arcuate nucleus insulin role in pain perception in streptozotocin (STZ)-induced diabetic and healthy rats. METHODS: Regular insulin was microinjected within the arcuate nucleus and the pain tolerance was measured using the hot plate and the tail-flick apparatus in diabetic rats. RESULTS: The results showed that the arcuate nucleus suppression with lidocaine could increase thermal nociception in non-diabetic animals. Also, insulin within the arcuate nucleus decreased the acute thermal pain perception in these animals. STZ-induced diabetes produced hypoalgesia which the latency of these tests, progressively increased over time after induction of diabetes. Also, in the same animal group, intra-arcuate injection of insulin reduced the latency of nociception. CONCLUSION: Intra-arcuate insulin has paradoxical and controversial effects in healthy and diabetic rats' nociception. These effects seem to be due to the insulin effect on releasing proopiomelanocortin and its derivatives.

Design, synthesis, in vivo and in vitro studies of 1,2,3,4-tetrahydro-9H-carbazole derivatives, highly selective and potent butyrylcholinesterase inhibitors.

Inhibition of butyrylcholinesterase (BChE) might be a useful therapeutic target for Alzheimer's disease (AD). A new series of 1,2,3,4-tetrahydro-9H-carbazole derivatives were designed synthesized and evaluated as BChE inhibitors. While all of the derivatives have shown for AChE IC50 values below the detectable limit (> 100 µM), they were selective potent BChE inhibitors. 1-(2-(6-fluoro-1,2,3,4-tetrahydro-9H-carbazole-9-yl)ethyl)piperidin-1-ium chloride (15 g) had the most potent anti-BChE activity (IC50 value = 0.11 µM), the highest BChE selectivity and mixed-type inhibition. Pharmacokinetic properties were accordant to Lipinski rule and compound 15g demonstrated neuroprotective and inhibition of ß-secretase (BACE1) activities. Furthermore, in vivo study of compound 15g in Morris water maze task has confirmed memory improvement in scopolamine-induced impairment. All results suggest that new sets of potent selective inhibitors of BChE have a therapeutic potential for the treatment of AD. A new series of 1,2,3,4-tetrahydro-9H-carbazole derivatives were designed synthesized and evaluated as BChE inhibitors. While all of the derivatives have shown for AChE IC50 values below the detectable limit, they were selective potent BChE inhibitors. Compound 15g had the most potent anti-BChE activity. All results suggest that new sets of potent selective inhibitors of BChE have a therapeutic potential for the treatment of AD.

Cannabinoid receptor modulation changes the accumbal neuronal responses to morphine in the reinstatement of morphine-induced conditioned place preference.

The nucleus accumbens (NAc) is a central component of the brain reward system. It has been known that most of the drugs of abuse such as opioids and cannabinoids affect the NAc. Although cannabinoids can modulate different stages of morphine encounter such as the reinstatement of morphine-induced conditioned place preference (CPP), there is no evidence for the NAc neurons' response to prove it. That is why the present study was designed. The procedure was as follows: The rats were entered to CPP by sc 5 mg/kg morphine in three consecutive days. During the extinction period or in the reinstatement phase, icv WIN 55,212-2 (10mM/5 µL dimethyl sulfoxide [DMSO] 10%) or AM251 (0.5mM/5-µL DMSO 10%) was infused in separate groups. Also, the NAc neurons' response to cannabinoid modulation in reinstatement to morphine was investigated by extracellular single unit recording. As a result, the cannabinoid in the reinstatement phase decreased the NAc neuronal activity. The CB1 receptor inhibition during the extinction period increased the NAc firing rate after ip 1 mg/kg morphine. Also, the inhibition of this receptor in the reinstatement phase increased the NAc neurons' firing rate. The inhibitory effect of cannabinoid on the NAc neuronal activity in the reinstatement has indicated the possible potency of cannabinoid to induce reinstatement of morphine-induced CPP alone and in the absence of a priming dose of morphine. Also, the different effects of the CB1 agonist during the extinction period in the reinstatement phase suggest different mechanisms underlying these two parts.

Design, synthesis, and biological evaluation of selective and potent Carbazole-based butyrylcholinesterase inhibitors.

Alzheimer's disease (AD) is the most common form of dementia. Inhibition of BChE might be a useful therapeutic target for AD. A new series of Carbazole-Benzyl Pyridine derivatives were designed synthesized and evaluated as butyrylcholinesterase (BChE) inhibitors. In vitro assay revealed that all of the derivatives had selective and potent anti- BChE activities. 3-((9H-Carbazol-9-yl)methyl)-1-(4-chlorobenzyl)pyridin-1-ium chloride (compound 8f) had the most potent anti-BChE activity (IC50 value = 0.073 µM), the highest BChE selectivity and mixed-type inhibition. Docking study revealed that 8f interacted with the peripheral site, the choline binding site, catalytic site and the acyl pocket of BChE. Physicochemical properties were accurate to Lipinski's rule. In addition, compound 8f demonstrated neuroprotective activity at 10 µM. This compound could also inhibit AChE-induced and self-induced Aß peptide aggregation at concentration of 100 µM and 10 µM respectively. The in-vivo study showed that compound 8f in 10 mg/kg increased the time spent in target quadrant in the probe day and decreased mean training period scape latency in rats. All results suggest that new sets of potent selective inhibitors of BChE have a therapeutic potential for the treatment of AD.

Modulatory role of the intra-accumbal CB1 receptor in protein level of the c-fos and pCREB/CREB ratio in the nucleus accumbens and ventral tegmental area in extinction and morphine seeking in the rats.

Brain reward and motivation circuit begin from the ventral tegmental area (VTA) that its dopaminergic terminals project to various regions of the brain including the nucleus accumbens (NAc). This reward circuit is influenced by drugs of abuse such as morphine and cannabinoid. The present study tried to investigate the role of the intra-accumbal CB1 receptor in the c-fos level and pCREB/CREB ratio in the NAc and the VTA during reinstatement phase of morphine-induced conditioned place preference (CPP) by western blotting. The present data reveals that intra-accumbal administration of CB1 agonist, WIN55,212-2 (0.5, 1 and 2 mM/0.5 µl DMSO) before/during extinction period of morphine-induced CPP, significantly decreased the NAc and the VTA c-fos protein level in the reinstatement phase; whereas the pre-reinstatement administration of the CB1 agonist, increased the c-fos protein level. Intra-accumbal administration of the CB1 agonist during the extinction period of morphine-induced CPP reduced the pCREB/CREB ratio in the NAc. Also, the present data show that intra-accumbal administration of CB1 antagonist, AM251 (15, 45 and 90 µM/0.5 µl DMSO) during/after extinction period of morphine-induced CPP affects the NAc and the VTA c-fos protein level in the reinstatement phase. Also, intra-NAc microinjection of AM251 during the extinction period reduced pCREB/CREB ratio in these regions. In conclusion, the results presented here provide compelling evidence of the modulation and involvement of the c-fos and the CREB molecules in the cannabinoid-opioid interaction of the brain reward system in the CPP paradigm.

Intra-accumbal Cannabinoid Agonist Attenuated Reinstatement but not Extinction Period of Morphine-Induced Conditioned Place Preference; Evidence for Different Characteristics of Extinction Period and Reinstatement.

The brain reward system consists of the ventral tegmental area that sends its dopaminergic projections to the forebrain, cortical areas, amygdala and largely to the nucleus accumbens (NAc). The present study aims were to investigate the effects of bilateral intra-accumbal microinjection of WIN55,212-2, a CB1 receptor agonist, on the duration of extinction period and reinstatement to morphine by the conditioned place preference (CPP) paradigm in the rat. Forty-six adult male albino Wistar rats received intra-accumbal WIN55,212-2 [p0.5, 1 and 2 mM/0.5 µl dimethyl sulfoxide (DMSO)] injections bilaterally. To induce CPP, morphine (5 mg/kg) was injected subcutaneously over three consecutive days. The results showed that intra-NAc administration of WIN55,212-2 during the extinction period had no effect on its duration but single administration of the1 mM/0.5 µl DMSO dose just before the reinstatement phase significantly attenuated its conditioning score. This is the first time that interactions of opioid and cannabinoid systems by local activation of CB1 receptors in the NAc during extinction and morphine-induced reinstatement were investigated. The CB1 agonist can inhibit and eliminate the reward-associated memory of morphine and the conditioning score in reinstatement but not in the extinction period. Our findings suggest that the extinction period and reinstatement could occur through different mechanisms.

Intra-accumbal CB1 receptor blockade reduced extinction and reinstatement of morphine.

The limbic dopaminergic reward system is the main target of morphine-like drugs which begins from the ventral tegmental area (VTA) and sends its dopaminergic projections to the nucleus accumbens (NAc), amygdala, hippocampus and prefrontal cortex. Cannabinoid receptors exist in afferent neurons from these areas to the NAc and can modulate glutamate synaptic transmission in the NAc. Cannabinoids can interact with the opiate system in reward-related behaviors; nevertheless these systems' interaction in extinction duration and reinstatement has not been shown. In the present study, the effects of bilateral intra-accumbal administration of AM251, a CB1 receptor antagonist, on the duration of the extinction phase and reinstatement to morphine were investigated by conditioned place preference (CPP) paradigm. Forty eight adult male albino Wistar rats were used. Bilateral intra-accumbal administration of AM251 (15, 45 and 90µM/0.5µl DMSO per side) was performed. Subcutaneous administration of morphine (5mg/kg) in three consecutive days was used to induce CPP. The results showed that administration of the maximal dose of AM251 during the extinction period significantly reduces duration of extinction and reinstatement to morphine. Administration of the middle dose during the extinction period significantly attenuated reinstatement to morphine. A single microinjection of the middle dose just before the reinstatement phase significantly attenuated reinstatement to morphine only, while bilateral intra-accumbal administration of neither the lowest dose nor the vehicle (DMSO) had any effects. These results for the first time indicated that CB1 receptors within the NAc are involved in the maintenance of morphine rewarding properties, and morphine seeking behaviors in extinguished morphine-induced CPP rats.