Chandrashekhar D. Borkar

A simple and inexpensive method to fabricate a cannula system for intracranial injections in rats and mice

INTRODUCTION Stereotaxic administration of neuroactive agents, either in ventricles, or targeted at specific intracranial sites, is a widely employed strategy for neurological studies in rodents. Surgical implantation of cannula on the skull is particularly useful in chronic treatments. We describe a simple, inexpensive and reliable method to fabricate a cannula system for delivery of drugs at the targeted sites in the brain of rat or mouse. METHODS The system consists of a guide cannula made from a hypodermic needle (24 gauge), a stainless steel wire (30 gauge) that serves as a dummy cannula, and an internal cannula made of stainless steel needle (30 gauge) taken from a hypodermic syringe. The cannula can be implanted by routine stereotaxic procedure and used for acute or chronic drug administration to conscious, free moving animals. RESULTS With a view to test the system for accuracy, the guide cannula was stereotaxically implanted, and neuropeptide Y was directly delivered into the lateral ventricle. These rats showed a significant increase in food intake. Another set of rats were cannulated for chronic protocol, wherein ethanol was delivered directly into the ventral tegmental area. In operant chamber, these rats showed increased ethanol self-administration. The proposed cannula takes around 5 min to fabricate and costs less than a dollar. CONCLUSION We feel that it may serve as an economical and reliable tool in neuropharmacological and neurobehavioral studies.

Differential expression of CART in feeding and reward circuits in binge eating rat model

Binge eating (BE) disrupts feeding and subverts reward mechanism. Since cocaine- and amphetamine-regulated transcript peptide (CART) mediates satiety as well as reward, its role in BE justifies investigation. To induce BE, rats were provided restricted access to high fat sweet palatable diet (HFSPD) for a period of 4 weeks. Immunoreactivity profile of the CART elements, and accompanying neuroplastic changes were studied in satiety- and reward-regulating brain nuclei. Further, we investigated the effects of CART, CART-antibody or rimonabant on the intake of normal chow or HFSPD. Rats fed on HFSPD showed development of BE-like phenotype as reflected by significant consumption of HFSPD in short time frame, suggestive of dysregulated satiety mechanisms. At the mid-point during BE, CART-immunoreactivity was significantly increased in hypothalamic arcuate (ARC), lateral (LH), nucleus accumbens shell (AcbSh) and paraventricular nucleus of thalamus (PVT). However, for next 22-h post-binge time-period, the animals showed no interest in food, and low CART expression. Pre-binge treatment with rimonabant, a drug recommended for the treatment of BE, produced anorexia, increased CART expression in ARC and LH, but not in AcbSh and PVT. Higher dose of CART was required to produce anorexia in binged rats. While neuronal tracing studies confirmed CART fiber connectivity from ARC and LH to AcbSh, increase in CART and synaptophysin immunostaining in this pathway in BE rats suggested strengthening of the CART connectivity. We conclude that CART bearing ARC-LH-PVT-AcbSh reward circuit may override the satiety signaling in ARC-PVN pathway in BE rats.

Neuropeptide Y attenuates anxiety- and depression-like effects of cholecystokinin-4 in mice

We investigated the involvement of neuropeptide Y (NPY) in the modulation of cholecystokinin-4 (CCK-4)-evoked anxiety and depression. Adult male mice were injected with vehicle, CCK-4, NPY, NPY Y1 receptor agonist [Leu(31), Pro(34)]-NPY or antagonist BIBP3226, via intracerebroventricular route, and subjected to social interaction or forced swim test (FST) for the evaluation of anxiety- and depression-like phenotypes, respectively. To assess the interactions between the two systems, if any, NPYergic agents were administered prior to CCK-4 and the animals were subjected to these behavioral tests. Treatment with CCK-4 or BIBP3226 dose-dependently reduced social interaction time, while NPY or [Leu(31), Pro(34)]-NPY produced opposite effect. CCK-4 treatment increased immobility time in FST. This effect was reversed by NPY and [Leu(31), Pro(34)]-NPY, although BIBP3226 per se did not alter the immobility time. In a combination study, the anxiogenic or depressive effects of CCK-4 were attenuated by NPY or [Leu(31), Pro(34)]-NPY and potentiated by BIBP3226. The brains of CCK-4 treated rats were processed for NPY immunohistochemistry. Following CCK-4 treatment, the nucleus accumbens shell (AcbSh), ventral part of lateral division of the bed nucleus of stria terminalis (BSTLV), hypothalamic paraventricular nucleus and locus coeruleus showed a reduction in NPY-immunoreactive fibers. Population of NPY-immunopositive cells was also decreased in the AcbSh, BSTLV, prefrontal cortex and hypothalamic arcuate nucleus (ARC). However, NPY-immunoreaction in the fibers of the ARC and cells of the central nucleus of amygdala was unchanged. We conclude that, inhibition of NPY signaling in the brain by CCK-4 might be causal to anxiety- and depression-like behaviors.

Neuropeptide Y system in accumbens shell mediates ethanol self‐administration in posterior ventral tegmental area

Although modulatory effects of neuropeptide Y (NPY) on ethanol consumption are well established, its role in ethanol reward, in the framework of mesolimbic dopaminergic system, has not been studied. We investigated the influence of nucleus accumbens shell (AcbSh) NPYergic system on ethanol self‐administration in posterior ventral tegmental area (p‐VTA) using intracranial self‐administration paradigm. Rats were stereotaxically implanted with cannulae targeted unilaterally at the right p‐VTA and trained to self‐administer ethanol (200 mg%) in standard two‐lever (active/inactive) operant chamber, an animal model with high predictive validity to test the rewarding mechanisms. Over a period of 7 days, these rats showed a significant increase in the number of lever presses for ethanol self‐administration suggesting reinforcement. While intra‐AcbSh NPY (1 or 2 ng/rat) or [Leu31, Pro34]‐NPY (0.5 or 1 ng/rat) dose‐dependently increased ethanol self‐administration, BIBP3226 (0.4 or 0.8 ng/rat) produced opposite effect. The rats conditioned to self‐administer ethanol showed significant increase in the population of NPY‐immunoreactive cells and fibres in the AcbSh, central nucleus of amygdala (CeA), hypothalamic arcuate nucleus (ARC) and lateral part of bed nucleus of stria terminalis as compared with that in the naïve rats. Neuronal tracing studies showed that NPY innervations in the AcbSh may derive from the neurons of ARC and CeA. As NPY and dopamine systems in reward areas are known to interact, we suggest that NPY inputs from ARC and CeA may play an important role in modulation of the dopaminergic system in the AcbSh and consequently influence the ethanol induced reward and addiction.

Pro‐cognitive action of CART is mediated via ERK in the hippocampus

Although cocaine‐ and amphetamine‐regulated transcript peptide (CART) is detected in several cortical and subcortical areas, its role in higher functions has been largely ignored. We examined the significance of CART in memory formation and tested if the downstream actions of CART involve N‐methyl‐d‐aspartate (NMDA) activated extra‐cellular signal‐regulated kinase (ERK). Newly formed memory was evaluated using novel object recognition test consisting of familiarization (T1) and choice trials (T2). The choice trials were performed at two time points: 30‐min (T230‐min) and 24‐h (T224‐h) postacquisition. In choice trial (T230‐min), vehicle control rats explored the novel object for significantly longer duration than the familiar object indicating intact memory formation. However, CART‐antibody, U0126 [ERK antagonist, both via intracerebroventricular (icv) or intrahippocampal (ih) route] or MK‐801 (NMDA antagonist; intraperitoneal) treated rats spent less time exploring novel objects; CART peptide (icv or ih) was ineffective. During choice trial at T224‐h, a significant decrease in novel object exploration time was noticed in vehicle control rats suggesting amnesia. However, treatment with CART, prior to familiarization trial (T1), promoted exploration of the novel object even at T224‐h. Pretreatment with U0126 or MK‐801 blocked pro‐cognitive‐like effect of CART suggesting involvement of NMDA‐ERK pathway in CARTs action. Animals subjected to the object familiarization trial showed a drastic increase in the CART‐immunoreactivity in the cells of cornu ammonis 3 and polymorph layer of dentate gyrus, and fibers within ento‐ (ENT) and peri‐rhinal (PRH) cortices. Western blot analysis revealed that CART treatment significantly up‐regulated the expression of phospo‐ERK1/2 in hippocampus, ENT and PRH. This effect was attenuated following pretreatment with U0126 or MK‐801, suggesting the activation of ERK signaling cascade through NMDA receptors. Thus, CART system seems to play an important role in recognition memory and that these effects may be mediated by NMDA receptors‐ERK signaling in the ENT/PRH‐hippocampal circuit.

Traumatic stress-induced persistent changes in DNA methylation regulate neuropeptide Y expression in rat jejunum

Stress‐induced chronic neuropsychiatric conditions such as anxiety are often co‐morbid with gastrointestinal malfunctions. While we find enduring anxiety‐like symptoms following minimal traumatic brain injury (MTBI) in rats, gastrointestinal consequences of MTBI remain elusive.

Cocaine- and Amphetamine-Regulated Transcript Peptide (CART) Alleviates MK-801-Induced Schizophrenic Dementia-Like Symptoms

Exaggerated thoughts, diminished mood and impaired cognition are the hallmarks of the schizophrenia-like condition. These symptoms are attributed to the dysregulation of dopamine and glutamate signaling in the brain. Since cocaine- and amphetamine-regulated transcript peptide (CART) modulates actions of dopamine as well as glutamate, we tested the role of this peptide in MK-801-induced schizophrenic dementia-like condition. MK-801-treated rats were allowed to interact with conspecific juvenile and tested for short-term (30-min) and long-term (24-h) social memory acquisition and recall. While MK-801 impaired the social interaction with a juvenile, the behavior was restored in CART [intracerebroventricular (icv) or intra-ventral tegmental area (VTA)] pre-treated animals. This action of CART was blocked by SCH23390 (dopamine D1 receptor antagonist) administered directly into the prefrontal cortex (PFC). Application of neuronal tracer Di-I in the PFC retrogradely labeled dopamine cells of the VTA, which in turn seem to receive CARTergic innervation. A significant increase in CARTimmunoreactivity was evidenced in the VTA, PFC and accumbens of the animals allowed to interact with a juvenile. However, MK-801 treatment attenuated the peptide expression and induced social memory deficits. The schizophrenic dementia-like symptoms following antagonism of glutamatergic receptors may be attributed to the reduced dopamine activity in the mesocortical system. We suggest that CART may, positively modulate the dopamine system to alleviate cognitive deficits associated with schizophrenia.

Neuropeptide CART prevents memory loss attributed to withdrawal of nicotine following chronic treatment in mice: CART in nicotine withdrawal

Although chronic nicotine administration does not affect memory, its withdrawal causes massive cognitive deficits. The underlying mechanisms, however, have not been understood. We test the role of cocaine‐ and amphetamine‐regulated transcript peptide (CART), a neuropeptide known for its procognitive properties, in this process. The mice on chronic nicotine treatment/withdrawal were subjected to novel object recognition task. The capability of the animal to discriminate between the novel and familiar objects was tested and represented as discrimination index (DI); reduction in the index suggested amnesia. Nicotine for 49 days had no effect on DI, but 8‐hour withdrawal caused a significant reduction, followed by full recovery at 24‐hour withdrawal timepoint. Bilateral CART infusion in dorsal hippocampus rescued deficits in DI at 8‐hours, whereas CART‐antibody infusion into the dorsal hippocampus attenuated the recovery at 24‐hours. Commensurate changes were observed in the CART as well as CART mRNA profiles in the hippocampus. CART mRNA expression and the peptide immunoreactivity did not change significantly following chronic nicotine treatment. However, there was a significant reduction at 8‐hour withdrawal, followed by a drastic increase in CART immunoreactivity as well as CART mRNA at 24‐hour withdrawal, compared with 8‐hour withdrawal. Distinct α7‐nicotinic receptor immunoreactivity was detected on the hippocampal CART neurons, suggesting cholinergic inputs. An increase in the synaptophysin immunoreactive elements around CART cells in the dentate gyrus, cornu ammonis 3 and subiculum at 24‐hour post‐withdrawal timepoint suggested neuronal plasticity. CART circuit dynamics in the hippocampus seems to modulate short‐term memory associated with nicotine withdrawal.