Browsing by Subject "DREADD"

Nucleus accumbens, located in ventral striatum, is an important part of the brain reward system. Accumbens integrates information coming from various brain areas, and it’s important for feeling pleasure, reward learning and reward seeking, including drugs of abuse. Cholinergic interneurons represent a few percent of accumbal cells. Earlier research suggests that accumbal cholinergic activity decreases drug seeking and eating. The aim of this study was to examine the role of cholinergic interneuron activity in alcohol drinking and alcohol related locomotor activity. Cholinergic interneurons (ChI) were manipulated using DREADDs (Designer Receptors Exclusively Activated by Designer Drugs), which can be selectively activated with clozapine-n-oxide (CNO). To express DREADDs selectively in ChIs, a cre-dependent viral vector that contained a gene coding for a cell-activating hM3D(Gq)-mCherry (n=9), cell-inhibiting hM4D(Gi)-mCherry (n=9), or control mCherry(n=8), was injected to nucleus accumbens of ChAT-cre- mice. Alcohol drinking was measured using Drinking In the Dark (DID)- model. Three hours after lights-out, the water bottles were replaced with 20% alcohol for two hours, for three days. On the fourth day, mice were injected with CNO or vehicle and alcohol was given for four hours. These cycles were repeated six times. In the locomotor assay, mice were injected with CNO or vehicle, followed by injection of alcohol or saline. Locomotor activity of the mice was observed for 30 minutes. In the DID- assay, the DREADD ligand CNO did not have effects on alcohol drinking within any of the three groups. However, Gi- mice drank more alcohol than Gq-mice even without the presence of CNO. These results are not reliable enough to draw conclusions, as they were confounded by unusually low drinking volumes. In the locomotor assay, CNO alone did not affect locomotion in any group. Together with alcohol, however, CNO decreased locomotion in all three groups, compared to alcohol alone. This is consistent with recent reports suggesting that CNO may have nonspecific behavioral effects.

Pedunculopontine tegmental nucleus (PPTg) has been connected to many different brain functions. Lesions in this region have been demonstrated to have effects on drug related behavior, learning and locomotion. Furthermore, glutamatergic projections to VTA have been found in earlier studies. VTA is one of the most important brain regions related to feeling of reward. Thus, it is plausible that glutamatergic cells are the ones that are responsible for these alterations in drug related behaviour. Additionally, there are efferents to basal ganglia and regions related to them, which indicates that PPTg is likely to possess some functions related to locomotion. The aim of this study was to explore what kind of differences in food consumption, spontaneous locomotion, morphine induced locomotion and morphine induced conditioned place preference result from modulation of activity of the glutamatergic neurons of the PPTg in mice. In this study DREADD-method was used. This method allowed for both excitation and inhibition of the neurons. This method involves injection viral vector stereotactically to the desired brain region, which leads to expression of artificial receptor in specific type of neurons. These receptors can then be activated by injecting clozapine-Noxide(CNO) intraperitoneally. There were two test groups, one got receptor that activates the neuron when activated, other got one that inhibits the neuron. Control group got receptor that does not react to CNO. In the tests conducted, there was no difference in the amount of food consumed or distance moved spontaneously. In the conditioned place preference experiment, there was no significant difference in distance moved between groups. However, both of the test groups expressed weaker preference to the morphine-paired environment when compared to the control group. This could be explained by reinforcing effects of morphine being mediated through glutamatergic neurons of the PPTg.

The bed nucleus of the stria terminalis (BNST) is currently widely studied due to its impact in the anxiety-, stress-, and fearrelated behaviours, as well as in addiction. The BNST is highly heterogeneous brain area constituting of set of subnuclei and a variety of neuron populations, properties of which have only partially been revealed by the earlier research. One of the neuron populations, on which only a very little research has been conducted, is the somatostatin (Sst) expressing neurons, highly abundant in the anterodorsal part of the BNST (adBNST), especially in oval and juxtacapsular nuclei of the BNST. This work aims to elucidate the connectivity of this Sst-neuron population, and their role in the behaviours related to BNST activation, particularly the anxiety-, reward-, and drug withdrawal-related behaviours. To specifically study the somatostatin neuron population in the adBNST, I targeted the neurons using stereotaxic delivery of AAV-vectors encoding a myristylated green fluorescent protein (GFP) for neuronal tracing to Sst-Cre-tdTomato reporter line mice (n=2), and Cre-inducible hM3Dq-DREADDs to Sst-IRES-Cre mice (n=21), with Cre-inducible mCherry fluorescent protein as a control (n=20). The mice were treated with activation-inducing 1.0 mg/kg i.p. clozapine-N-oxide (CNO) 30 min prior to the behavioural tests. To assess acute anxiety-like behaviour, I used the elevated-plus maze paradigm and a modified open field test, in which a novel object is introduced to the arena in the middle of the trial. To study the potential effect on reward-associated behaviours, I used the biased conditioned place preference (CPP) test, and for the withdrawal-linked behaviours, we used a method to precipitate the withdrawal symptoms with naltrexone in subchronically morphine-treated mice (n=9 hM3Dq, n=8 control). The neuronal tracing revealed that the adBNST Sst-neurons project to areas known to partake in stress and fear reactions as well as in autonomic and homeostatic control. Namely, projections were seen in medial and central amygdaloidal nuclei, lateral hypothalamus, periaqueductal grey, ventral pallidum, and parabrachial nucleus. In the elevated-plus maze, the CNO-induced activation of the Sst-neurons did not have any effect on the locomotor activity of the mice between the groups. At the same time, Sst activation did not seem to have any significant effect on the time the mice spent in the open arms, nor in the exploratory activities, like the frequency of the head dips or the stretch-attend postures. In line with these results, no effect on the movement between the groups was observed in the open field test. Similarly, no differences in anxiety-related behaviours, like in the time spent in the centre of the arena or in the number of contacts with the novel object during the last phase of the test, were observed. The CPP test failed to show any meaningful rewarding or aversive properties of CNO-induced activation of the Sst-neurons, while the movement rates of the groups during the conditioning trials were not different in statistically significant way. As for the withdrawal symptoms, all the mice showed the predetermined symptoms, but the test failed to show any differences between the study groups. The neuronal tracing revealed connectivity for the adBNST Sst-neurons with brain regions involved in fear- and anxiety behaviour, social encounters, and autonomic control. In spite of this, the CNO-induced chemogenetic activation of the adBNST Sst-neurons failed to show any significant behavioural effects in the chosen paradigms for anxiety-, and reward-related behaviours, and for withdrawal symptoms. Further research is needed to dissect the Sst-subcircuitry of adBNST, both in order to verify the observed output regions, and to elucidate the role these neurons play in modification of behavioural phenotypes.