Gajanan P. Shelkar

Agmatine, an endogenous imidazoline receptor ligand modulates ethanol anxiolysis and withdrawal anxiety in rats.

Present study investigated the role of agmatine in ethanol-induced anxiolysis and withdrawal anxiety using elevated plus maze (EPM) test in rats. The anxiolytic-like effect of ethanol was potentiated by pretreatment with imidazoline I(1)/I(2) receptor agonist agmatine (10-20 mg/kg, i.p.), imidazoline I(1) receptor agonists, moxonidine (0.25 mg/kg, i.p.) and clonidine (0.015 mg/kg, i.p.), imidazoline I(2) receptor agonist, 2-BFI (5 mg/kg, i.p.) as well as by the drugs known to increase endogenous agmatine levels in brain viz., L-arginine, an agmatine biosynthetic precursor (100 microg/rat, i.c.v.), ornithine decarboxylase inhibitor, DFMO (125 microg/rat, i.c.v.), diamine oxidase inhibitor, aminoguanidine (65 microg/rat, i.c.v.) and agmatinase inhibitor, arcaine (50 microg/rat, i.c.v.). Conversely, prior administration of I(1) receptor antagonist, efaroxan (1 mg/kg, i.p.), I(2) receptor antagonist, idazoxan (0.25mg/kg, i.p.) and arginine decarboxylase inhibitor, D-arginine (100 microg/rat, i.c.v.) blocked the anxiolytic-like effect of ethanol. Moreover, ethanol withdrawal anxiety was markedly attenuated by agmatine (10-20 mg/kg, i.p.), moxonidine (0.25 mg/kg, i.p.), clonidine (0.015 mg/kg, i.p.), 2-BFI (5 mg/kg, i.p.), L-arginine (100 microg/rat, i.c.v.), DFMO (125 microg/rat, i.c.v.), aminoguanidine (65 microg/rat, i.c.v.) and arcaine (50 microg/rat, i.c.v.). The anti-anxiety effect of agmatine in ethanol-withdrawn rats was completely blocked by efaroxan (1 mg/kg, i.p.) and idazoxan (0.25 mg/kg, i.p.). These results suggest that agmatine and imidazoline receptor system may be implicated in ethanol-induced anxiolysis and withdrawal anxiety and strongly support further investigation of agmatine in ethanol dependence mechanism. The data also project agmatine as a potential therapeutic target in overcoming alcohol withdrawal symptoms such as anxiety.

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.

Alpha-melanocyte stimulating hormone modulates ethanol self-administration in posterior ventral tegmental area through melanocortin-4 receptors

Although the role of alpha‐melanocyte stimulating hormone (α‐MSH) in alcohol seeking behaviour in rats has been demonstrated, the underlying mechanisms are not understood. Herein, we test the hypothesis that α‐MSH might have a permissive effect in promoting the reward action of ethanol. Rats were implanted with cannulae targeted at the posterior ventral tegmental area (pVTA), because the site is sensitive to reinforcing effects of ethanol. These rats were trained to self‐administer ethanol in standard two‐lever (active/inactive) operant chamber test. Each active lever press resulted in self‐administration of 100 nl of ethanol (100–300 mg%) containing solution. Over a period of 7 days, ethanol significantly increased the number of lever presses, which was considered as a measure of reward. Because ethanol at 200 mg% resulted in maximum number of lever presses (∼18–20 lever presses/30‐minute session), the dose was employed in further studies. While prior administration of melanocortin (MC) agonists, α‐MSH or [Nle4,D‐Phe7]‐alpha‐MSH into pVTA, resulted in an 89% increase in lever presses, the response was attenuated following pre‐treatment with MC4 receptors (MC4R) antagonist, HS014. In an immunohistochemical study, the brains of rats that were trained to self‐infuse ethanol showed significantly increased α‐MSH immunoreactivity in the nucleus accumbens shell, bed nucleus of stria terminalis and arcuate nucleus of the hypothalamus. In the pVTA, α‐MSH fibres were found to run close to the dopamine cells, labelled with tyrosine hydroxylase antibodies. We suggest that α‐MSH‐MC4R system in the pVTA might be a part of the neuroadaptive mechanism underlying ethanol addiction.

Agmatine attenuates nicotine induced conditioned place preference in mice through modulation of neuropeptide Y system

The purpose of the present study was to examine the effect of agmatine on nicotine induced conditioned place preference (CPP) in male albino mice. Intra-peritoneal (ip) administration of nicotine (1mg/kg) significantly increased time spent in drug-paired compartment. Agmatine (20 and 40 mg/kg, ip) co-administered with nicotine during the 6 days conditioning sessions completely abolished the acquisition of nicotine-induced CPP in mice. Concomitant administration of neuropeptide Y (NPY) (1 pg/mouse, icv) or [Leu(31), Pro(34)]-NPY (0.1 pg/mouse, icv), selective NPY Y1 receptor agonist potentiated the inhibitory effect of agmatine (10 mg/kg, ip) on nicotine CPP. Conversely, pretreatment with NPY Y1 receptor antagonist, BIBP3226 (0.01 ng/mouse, icv) blocked the effect of agmatine (20 mg/kg, ip) on nicotine induced CPP. In immunohistochemical study, nicotine decreased NPY-immunoreactivity in nucleus accumbens shell (AcbSh), bed nucleus of stria terminalis, lateral part (BNSTl), arcuate nucleus (ARC) and paraventricular nucleus (PVN). Conversely, administration of agmatine prior to the nicotine significantly reversed the effect of nicotine on NPY-immunoreactivity in the above brain nuclei. This data indicate that agmatine attenuate nicotine induced CPP via modulation of NPYergic neurotransmission in brain.

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.

The role of neuropeptide CART in the lateral hypothalamic-ventral tegmental area (LH-VTA) circuit in motivation

HighlightsIntracranial self‐stimulation (ICSS) was facilitated by CART.ICSS resulted in robust activation of CART cells in the LH.ICSS induced synaptic plasticity in CART system in reward circuit.Rats can be conditioned to self‐administer CART in pVTA.CART neurons of LH‐MFB may process motivation via the mesolimbic dopamine pathway. ABSTRACT Rats with electrode implanted in the lateral hypothalamus (LH)‐medial forebrain bundle (MFB) area actively engage in intracranial self‐stimulation (ICSS). However, the neuronal substrate that translates the electrical pulses into the neural signals, and integrates the information with mesolimbic reward system, has remained elusive. We test the hypothesis that the cocaine‐ and amphetamine‐regulated transcript (CART) neurons in the LH‐MFB area may support this function. The ICSS activity via an electrode in LH‐MFB area was facilitated by CART (55–102) peptide stereotaxically injected in the lateral ventricle or posterior ventral tegmental area (pVTA), but attenuated by CART antibody. While the ICSS experience seems to activate CART cells in the LH, the pVTA showed significant increment in the CART fiber terminals on the dopamine cells, increase in tyrosine hydroxylase (TH)‐immunoreactivity, and CART and synaptophysin colabeled elements. Neuronal tracing experiments revealed that CART cells of the LH‐MFB region project to the pVTA. The rats with stereotaxically implanted cannulae in pVTA avidly self‐infused CART (55–102) suggesting a role for the peptide in motivation, however, CART (1–39) was ineffective. CART self‐infusing activity was inhibited by dopamine D1 receptors antagonist, given directly in the nucleus accumbens shell (AcbSh). The rats trained to self‐administer CART (55–102) showed enhanced TH immunoreactivity in the cells of pVTA and fibers in AcbSh. We suggest that CART neurons of the LH‐MFB area may play a role in conveying reward information to the mesolimbic dopamine neurons, which in turn may arouse the goal directed behavior.

Minimal traumatic brain injury causes persistent changes in DNA methylation at BDNF gene promoters in rat amygdala: A possible role in anxiety-like behaviors

Minimal traumatic brain injury (MTBI) often transforms into chronic neuropsychiatric conditions including anxiety, the underlying mechanisms of which are largely unknown. In the present study, we employed the closed-head injury paradigm to induce MTBI in rats and examined whether DNA methylation can explain long-term changes in the expression of the brain-derived neurotrophic factor (BDNF) in the amygdala as well as trauma-induced anxiety-like behaviors. The MTBI caused anxiety-like behaviors and altered the expression of DNA methyltransferase (DNMT) isoforms (DNMT1, DNMT3a, and DNMT3b) and factors involved in DNA demethylation such as the growth arrest and DNA damage 45 (GADD45a and GADD45b). After 30days of MTBI, the over-expression of DNMT3a and DNMT3b corresponded to heightened DNMT activity, whereas the mRNA levels of GADD45a and GADD45b were declined. The methylated cytosine levels at the BDNF promoters (Ip, IVp and IXp) were increased in the amygdala of the trauma-induced animals; these coincided negatively with the mRNA levels of exon IV and IXa, but not of exon I. Interestingly, treatment with 5-azacytidine, a pan DNMT inhibitor, normalized the MTBI-induced DNMT activity and DNA hypermethylation at exon IVp and IXp. Furthermore, 5-azacytidine also corrected the deficits in the expression of exons IV and IXa and reduced the anxiety-like behaviors. These results suggest that the DNMT-mediated DNA methylation at the BDNF IVp and IXp might be involved in the regulation of BDNF gene expression in the amygdala. Further, it could also be related to MTBI-induced anxiety-like behaviors via the regulation of synaptic plasticity.

Noradrenergic inputs from locus coeruleus to posterior ventral tegmental area are essential to support ethanol reinforcement.

Although dysregulation of the dopaminergic mesolimbic system is generally considered central to addiction, the involvement of other circuits is increasingly being appreciated. An interaction between locus coeruleus (LC) noradrenergic neurons and the posterior ventral tegmental area (pVTA) dopaminergic system, in the processing of drug‐triggered reward, has been suggested, but not demonstrated in behaving animals. Herein, we try to tease out the precise role of noradrenergic neurons in the LC–VTA circuit in mediating reward and reinforcement behavior associated with ethanol. In the standard two‐lever (active/inactive) operant paradigm, the rats were trained to self‐administer ethanol in pVTA and subjected to pharmacological intervention. Intra‐pVTA administration of phenylephrine (alpha‐1 adrenoceptor agonist) increased ethanol self‐administration, while prazosin and disulfiram (agents that reduce noradrenergic tone) produced opposite effects. While degeneration [N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine hydrochloride, DSP‐4, intraperitoneal route] or silencing (lidocaine or muscimol, both via intra‐LC route) of the LC noradrenergic neurons decreased, phenylephrine via the intra‐LC route reinstated ethanol self‐administration. Furthermore, lidocaine reduced ethanol self‐administration, but the effect was fully attenuated by noradrenaline given directly in the pVTA. This suggests that the feedback signals from LC to pVTA are necessary to sustain the ethanol self‐infusion activity. Ethanol self‐administration significantly increased tyrosine hydroxylase immunoreactivity in pVTA and LC; the response was blocked by DSP‐4 pre‐treatment. While dopamine D1, but not D2, receptors were localized on noradrenergic LC neurons, pre‐treatment with SCH‐23390 (intra‐LC) dampened the lever press activity. We suggest that two‐way communications between VTA and LC regions is essential for ethanol‐triggered reinforcement behavior.

Nitric Oxide‐α2‐adrenergic receptor interaction within locus coeruleus underlies facilitation of inhibitory avoidance memory by agmatine

Agmatine, a putative neurotransmitter, plays a vital role in learning and memory. Although it is considered an endogenous ligand of imidazoline receptors, agmatine exhibits high affinity for α‐adrenoceptors, NOS and NMDA receptors. These substrates within the locus coeruleus (LC) are critically involved in learning and memory processes.

Interactions of nitric oxide with α2-adrenoceptors within the locus coeruleus underlie the facilitation of inhibitory avoidance memory by agmatine

Agmatine, a putative neurotransmitter, plays a vital role in learning and memory. Although it is considered an endogenous ligand of imidazoline receptors, agmatine exhibits high affinity for α‐adrenoceptors, NOS and NMDA receptors. These substrates within the locus coeruleus (LC) are critically involved in learning and memory processes.