Societal costs of illegal drug use in Sweden.

BACKGROUND: Illegal drug use is a public health concern with far-reaching consequences for people who use them and for society. In Sweden, the reported use of illegal drugs has been growing and the number of drug-induced deaths is among the highest in Europe. The aim of this study was to provide a comprehensive and up-to-date estimation of the societal costs of illegal drug use in Sweden, relying as much as possible on registry and administrative data. METHODS: A prevalence-based cost-of-illness study of illegal drug use in Sweden in 2020 was conducted. A societal approach was chosen and included direct costs (such as costs of health care, social services, and the criminal justice system), indirect costs (such as lost productivity due to unemployment and drug-induced death), and intangible costs (such as reduced quality of life among people who use drugs and their family members). Costs were estimated by combining registry, administrative, and survey data with unit cost data. RESULTS: The estimated societal costs of illegal drug use were 3.7 billion euros in 2020. This corresponded to 355 euros per capita and 0.78 % of the gross domestic product. The direct and intangible costs were of similar sizes, each contributing to approximately 40 % of total costs, whereas indirect costs contributed to approximately 20 %. The largest individual cost components were reduced quality of life among people who use drugs and costs of the criminal justice system. CONCLUSION: Illegal drug use has a negative impact on the societal aim to create good and equitable health in Sweden. The findings call for evidence-based prevention of drug use and treatment for those addicted. It is important to address the co-morbidity of mental ill-health and drug dependence, to develop low-threshold services and measures for early prevention among children and young adults, as well as to evaluate laws and regulations connected to illegal drug use.

Using in vitro receptor activity studies of synthetic cannabinoids to support the risk assessment of new psychoactive substances - A Swedish strategy to protect public health from harm.

In the past 15 years, close to 1000 of new psychoactive substances (NPS) have been reported in Europe and globally. At the time of identification, data on safety, toxicity and carcinogenic potential of many NPS are not available or very limited. To work more efficiently, a strategy and collaboration between the Public Health Agency of Sweden (PHAS) and the National Board of Forensic Medicine was established involving in vitro receptor activity assays to demonstrate neurological activity of NPS. This report summarizes the first results on the synthetic cannabinoid receptor agonists (SCRAs), and subsequent actions taken by PHAS. A total of 18 potential SCRAs were selected by PHAS for in vitro pharmacological characterization. 17 compounds could be acquired and investigated for their activity on the human cannabinoid-1 (CB1) receptors expressed together with the AequoScreen system in CHO-K1 cells. Dose-response curves were established using eight different concentrations in triplicates at three occasions with JWH-018 as reference. For the MDMB-4en-PINACA, MMB-022, ACHMINACA, ADB-BUTINACA, 5F-CUMYL-PeGACLONE, 5C-AKB48, NM-2201, 5F-CUMYL-PINACA, JWH-022, 5Cl-AB-PINACA, MPhP-2201, 5F-AKB57 the half maximal effective concentration values ranged from 2.2 nM (5F-CUMYL-PINACA) to 171 nM (MMB-022). EG-018 and 3,5-AB-CHMFUPPYCA were none-active. The results contributed to 14 of these compounds being scheduled as narcotics in Sweden. In conclusion, many of the emerging SCRAs are potent activators of the CB1 receptor in vitro, although some lack activity or are partial agonists. The new strategy proved useful when data on psychoactive effects of the SCRAs under investigation were not available or limited.

Upregulation of the Endogenous Peptide Antisecretory Factor Enhances Hippocampal Long-term Potentiation and Promotes Learning in Wistar Rats.

Antisecretory Factor (AF) is an endogenous peptide known for its powerful antisecretory and anti-inflammatory properties. We have previously shown that AF also acts as a neuromodulator of GABAergic synaptic transmission in rat hippocampus in a way that results in disinhibition of CA1 pyramidal neurons. Disinhibition is expected to facilitate the induction of long-term potentiation (LTP), and LTP is known to play a crucial role in learning and memory acquisition. In the present study we investigated the effect of AF on LTP in CA3-CA1 synapses in rat hippocampus. In addition, endogenous AF plasma activity was upregulated by feeding the rats with specially processed cereals (SPC) and spatial learning and memory was studied in the Morris Water Maze (MWM). We found that LTP was significantly enhanced in the presence of AF, both when added exogenously in vitro as well as when upregulated endogenously by SPC-feeding. In the presence of the GABAA-receptor antagonist picrotoxin (PTX) there was however no significant enhancement of LTP. Moreover, rats fed with SPC demonstrated enhanced spatial learning and short-term memory, compared with control animals. These results show that the disinhibition of GABAergic transmission in the hippocampus by the endogenous peptide AF enhances LTP as well as spatial learning and memory.

TGF-ß1 Suppresses Proliferation and Induces Differentiation in Human iPSC Neural in vitro Models.

Persistent neural stem cell (NSC) proliferation is, among others, a hallmark of immaturity in human induced pluripotent stem cell (hiPSC)-based neural models. TGF-ß1 is known to regulate NSCs in vivo during embryonic development in rodents. Here we examined the role of TGF-ß1 as a potential candidate to promote in vitro differentiation of hiPSCs-derived NSCs and maturation of neuronal progenies. We present that TGF-ß1 is specifically present in early phases of human fetal brain development. We applied confocal imaging and electrophysiological assessment in hiPSC-NSC and 3D neural in vitro models and demonstrate that TGF-ß1 is a signaling protein, which specifically suppresses proliferation, enhances neuronal and glial differentiation, without effecting neuronal maturation. Moreover, we demonstrate that TGF-ß1 is equally efficient in enhancing neuronal differentiation of human NSCs as an artificial synthetic small molecule. The presented approach provides a proof-of-concept to replace artificial small molecules with more physiological signaling factors, which paves the way to improve the physiological relevance of human neural developmental in vitro models.

Progressive modulation of accumbal neurotransmission and anxiety-like behavior following protracted nicotine withdrawal.

Due to the highly addictive properties of nicotine, a low percentage of users successfully maintain cessation for longer periods of time. This might be linked to neuroadaptations elicited by the drug, and understanding progressive changes in neuronal function might provide critical insight into nicotine addiction. We have previously shown that neurotransmission in the nucleus accumbens (nAc), a key brain region with respect to drug reinforcement and relapse, is suppressed for as long as seven months after a brief period of nicotine treatment. Studies were therefore performed to define the temporal properties of these effects, and to assess behavioral correlates to altered neurotransmission. Ex vivo electrophysiology revealed progressive depression of synaptic efficacy in the nAc of rats previously receiving nicotine. In addition, following three months of nicotine withdrawal, the responses to GABAA receptor modulating drugs were blunted together with downregulation of several GABAA receptor subunits. In correlation to reduced accumbal neurotransmission, a reduced anxiety-like behavior; assessed in the elevated plus-maze and marble burying tests, were identified in animals pre-treated with nicotine. Lastly, to test the causal relationship between suppressed excitability in the nAc and reduced anxiety-like behavior, rats received local administration of diazepam in the nAc while monitoring behavioral effects on the elevated plus-maze. These results show that nicotine produces long-lasting changes in the GABAergic system, which are observed first after extended withdrawal. Our data also suggest that nicotine produces a progressive suppression of accumbal excitability, which could result in behavioral alterations that may have implications for further drug intake.

Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation.

Amyloid precursor protein (APP) and its cleavage product amyloid ß (Aß) have been thoroughly studied in Alzheimer's disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while ß-cleaved soluble APP (sAPPß) was first secreted after deep-layer neurons had formed. Short Aß peptides, including Aß1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aß1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aß1-40/42, is associated with mature neuronal phenotypes.

The endogenous peptide antisecretory factor promotes tonic GABAergic signaling in CA1 stratum radiatum interneurons.

Tonic GABAergic inhibition regulates neuronal excitability and has been implicated to be involved in both neurological and psychiatric diseases. We have previously shown that the endogenous peptide antisecretory factor (AF) decreases phasic GABAergic inhibition onto pyramidal CA1 neurons. In the present study, using whole-cell patch-clamp recordings, we investigated the mechanisms behind this disinhibition of CA1 pyramidal neurons by AF. We found that application of AF to acute rat hippocampal slices resulted in a reduction of the frequency, but not of the amplitude, of spontaneous inhibitory postsynaptic currents (sIPSCs) in CA1 pyramidal neurons. Miniature inhibitory postsynaptic currents (mIPSCs), recorded in the presence of tetrodotoxin (TTX), were however not affected by AF, neither in CA1 pyramidal cells, nor in stratum radiatum interneurons. Instead, AF caused an increase of the tonic GABAA current in stratum radiatum interneurons, leaving the tonic GABAergic transmission in CA1 pyramidal cells unaffected. These results show that the endogenous peptide AF enhances tonic, but not phasic, GABAergic signaling in CA1 stratum radiatum interneurons, without affecting tonic GABAergic signaling in CA1 pyramidal neurons. We suggest that this increased tonic GABAergic signaling in GABAergic interneurons could be a mechanism for the AF-mediated disinhibition of pyramidal neurons.

AMPA receptor activation causes silencing of AMPA receptor-mediated synaptic transmission in the developing hippocampus.

Agonist-induced internalization of transmembrane receptors is a widespread biological phenomenon that also may serve as a mechanism for synaptic plasticity. Here we show that the agonist AMPA causes a depression of AMPA receptor (AMPAR) signaling at glutamate synapses in the CA1 region of the hippocampus in slices from developing, but not from mature, rats. This developmentally restricted agonist-induced synaptic depression is expressed as a total loss of AMPAR signaling, without affecting NMDA receptor (NMDAR) signaling, in a large proportion of the developing synapses, thus creating AMPAR silent synapses. The AMPA-induced AMPAR silencing is induced independently of activation of mGluRs and NMDARs, and it mimics and occludes stimulus-induced depression, suggesting that this latter form of synaptic plasticity is expressed as agonist-induced removal of AMPARs. Induction of long-term potentiation (LTP) rendered the developing synapses resistant to the AMPA-induced depression, indicating that LTP contributes to the maturation-related increased stability of these synapses. Our study shows that agonist binding to AMPARs is a sufficient triggering stimulus for the creation of AMPAR silent synapses at developing glutamate synapses.

Lamotrigine increases the number of BrdU-labeled cells in the rat hippocampus.

Antidepressant medication and electroconvulsive therapy stabilize mood symptoms and increase hippocampal neurogenesis. We examined whether lamotrigine, suggested to give rise to mood-stabilizing and antidepressant effects in addition to its antiepileptic properties, also increases the number of newborn cells in rat hippocampus. Rats (on day P21) received lamotrigine, valproate, or saline intraperitoneally once daily for 7 days. All animals received four intraperitoneal injections of bromodeoxyuridine (BrdU) on day P28 and were sacrificed the next day. Quantification of BrdU-labeled cells in the granule cell layer of the dentate gyrus showed an increased number of newborn cells in rats receiving lamotrigine (42.6 ± 3.5 cells/slice) compared with valproate (31.6 ± 2.8) and controls (32.2 ± 3.1; P<0.05). The increased number of BrdU-labeled cells suggests increased neurogenesis, possibly explaining the mood-stabilizing and antidepressant effects of lamotrigine.

Adult neural stem/progenitor cells reduce NMDA-induced excitotoxicity via the novel neuroprotective peptide pentinin.

Although the potential of adult neural stem cells to repair damage via cell replacement has been widely reported, the ability of endogenous stem cells to positively modulate damage is less well studied. We investigated whether medium conditioned by adult hippocampal stem/progenitor cells altered the extent of excitotoxic cell death in hippocampal slice cultures. Conditioned medium significantly reduced cell death following 24 h of exposure to 10 microM NMDA. Neuroprotection was greater in the dentate gyrus, a region neighboring the subgranular zone where stem/progenitor cells reside compared with pyramidal cells of the cornis ammonis. Using mass spectrometric analysis of the conditioned medium, we identified a pentameric peptide fragment that corresponded to residues 26-30 of the insulin B chain which we termed 'pentinin'. The peptide is a putative breakdown product of insulin, a constituent of the culture medium, and may be produced by insulin-degrading enzyme, an enzyme expressed by the stem/progenitor cells. In the presence of 100 pM of synthetic pentinin, the number of mature and immature neurons killed by NMDA-induced toxicity was significantly reduced in the dentate gyrus. These data suggest that progenitors in the subgranular zone may convert exogenous insulin into a peptide capable of protecting neighboring neurons from excitotoxic injury.

Modulation of low-frequency-induced synaptic depression in the developing CA3-CA1 hippocampal synapses by NMDA and metabotropic glutamate receptor activation.

Brief test-pulse stimulation (0.2-0.05 Hz) of naïve (previously nonstimulated) developing hippocampal CA3-CA1 synapses leads to a substantial synaptic depression, explained by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) silencing. Using field recordings in hippocampal slices from P8 to P12 rats, we examined this depression of naïve synapses using more prolonged test-pulse stimulation as well as low-frequency (1 Hz) stimulation (LFS). We found that 900 stimuli produced depression during stimulation to approximately 40% of the naïve level independent of whether test-pulse stimulation or LFS was used. This result was also observed during combined blockade of N-methyl-d-aspartate/metabotropic glutamate receptors (NMDAR/mGluRs) although the depression was smaller (to approximately 55% of naïve level). Using separate blockade of either NMDARs or mGluRs, we found that this impairment of the depression resulted from the NMDAR, and not from the mGluR, blockade. In fact, during NMDAR blockade alone, depression was smaller even than that observed during combined blockade. We also found that mGluR blockade alone facilitated the LFS-induced depression. In conclusion, test-pulse stimulation produced as much depression as LFS when applied to naïve synapses even when allowing for NMDAR and mGluR activation. Our results seem in line with the notion that NMDARs and mGluRs may exert a bidirectional control on AMPA receptor recruitment to synapses.

Restricted cortical and amygdaloid removal of vesicular glutamate transporter 2 in preadolescent mice impacts dopaminergic activity and neuronal circuitry of higher brain function.

A major challenge in neuroscience is to resolve the connection between gene functionality, neuronal circuits, and behavior. Most, if not all, neuronal circuits of the adult brain contain a glutamatergic component, the nature of which has been difficult to assess because of the vast cellular abundance of glutamate. In this study, we wanted to determine the role of a restricted subpopulation of glutamatergic neurons within the forebrain, the Vglut2-expressing neurons, in neuronal circuitry of higher brain function. Vglut2 expression was selectively deleted in the cortex, hippocampus, and amygdala of preadolescent mice, which resulted in increased locomotor activity, altered social dominance and risk assessment, decreased sensorimotor gating, and impaired long-term spatial memory. Presynaptic VGLUT2-positive terminals were lost in the cortex, striatum, nucleus accumbens, and hippocampus, and a downstream effect on dopamine binding site availability in the striatum was evident. A connection between the induced late-onset, chronic reduction of glutamatergic neurotransmission and dopamine signaling within the circuitry was further substantiated by a partial attenuation of the deficits in sensorimotor gating by the dopamine-stabilizing antipsychotic drug aripiprazole and an increased sensitivity to amphetamine. Somewhat surprisingly, given the restricted expression of Vglut2 in regions responsible for higher brain function, our analyses show that VGLUT2-mediated neurotransmission is required for certain aspects of cognitive, emotional, and social behavior. The present study provides support for the existence of a neurocircuitry that connects changes in VGLUT2-mediated neurotransmission to alterations in the dopaminergic system with schizophrenia-like behavioral deficits as a major outcome.

Ketogenic diet does not disturb neurogenesis in the dentate gyrus in rats.

The ketogenic diet, a high-fat diet, is a therapeutic alternative in the treatment of refractory epilepsy, especially in children. However, there are concerns that a high-fat diet may influence the normal development of the central nervous system and cognition. In this study we investigated the influence of ketogenic diet on adult neurogenesis in the dentate gyrus. Rats were provided with either a high-fat diet (80% fat) or a standard rat diet (5% fat) ad libitum for 4 weeks. In both female and male rats, the amounts of bromodeoxyuridine immunoreactive cells in the dentate gyrus were the same in the different groups. Our results suggest that the ketogenic diet does not disturb the neurogenesis in the rat dentate gyrus.

Evaluation of the role of abstinence in heroin overdose deaths using segmental hair analysis.

In the body heroin is rapidly metabolized to 6-acetylmorphine and morphine. Victims of lethal heroin overdose often present with fairly low blood concentrations of morphine. Reduced tolerance due to abstinence has been proposed to account for this finding. The aim of the present study was to examine the role of abstinence in drug-related deaths by comparing recent and past exposure to opioids using segmental hair analysis with the postmortem blood morphine concentrations in deceased heroin users. The study included 60 deceased drug addicts in the Stockholm area, Sweden. In 32 cases, death was not related to heroin intake. In 18 of the 28 heroin fatalities, opioids were absent in the most recent hair segment, suggesting a reduced tolerance to opioids. However, the blood morphine levels were similar to those found in the 10 subjects that showed continuous opioid use. Hair and blood analysis disclosed an extensive use of additional drugs that directly or indirectly may influence the opioid system. The results suggest that abstinence is not a critical factor for heroin overdose death. Obviously tolerant subjects die after intake of similar doses. Other factors, particularly polydrug use, seem to be more causally important for these deaths.

Tonic GABA(A) receptor-mediated currents in human brain.

GABA(A) receptors can mediate both phasic (synaptic) and tonic (extrasynaptic) forms of inhibition. It has been proposed that tonic inhibition plays a critical part in controlling neuronal and network excitability. Although tonic GABA(A) receptor-mediated currents have been well characterized in rodents, their existence in human tissue has yet to be demonstrated. Here we show that tonic currents can be recorded from human tissue obtained from patients undergoing temporal lobectomies. Tonic GABA(A) receptor-mediated currents were present in pyramidal cells and interneurons in layer V-VI of temporal neocortex and granule cells in the dentate gyrus. These tonic currents have cell type-specific pharmacologies, opening up the possibility of targeted therapeutics.

Toxicological analysis in rats subjected to heroin and morphine overdose.

In heroin overdose deaths the blood morphine concentration varies substantially. To explore possible pharmacokinetic explanations for variable sensitivity to opiate toxicity we studied mortality and drug concentrations in male Sprague-Dawley rats. Groups of rats were injected intravenously (i.v.) with heroin, 21.5 mg/kg, or morphine, 223 mg/kg, causing a 60-80% mortality among drug-naïve rats. Additional groups of rats were pre-treated with morphine for 14 days, with or without 1 week of subsequent abstinence. Brain, lung and blood samples were analyzed for 6-acetylmorphine, morphine, morphine-3-glucuronide and morphine-6-glucuronide. i.v. morphine administration to drug-naïve rats resulted in both rapid and delayed deaths. The brain morphine concentration conformed to an exponential elimination curve in all samples, ruling out accumulation of morphine as an explanation for delayed deaths. This study found no support for formation of toxic concentration of morphine-6-glucuronide. Spontaneous death among both heroin and morphine rats occurred at fairly uniform brain morphine concentrations. Morphine pre-treatment significantly reduced mortality upon i.v. morphine injection, but the protective effect was less evident upon i.v. heroin challenge. The morphine pre-treatment still afforded some protection after 1 week of abstinence among rats receiving i.v. morphine, whereas rats given i.v. heroin showed similar death rate as drug-naïve rats.

Screening for drugs of abuse in hair with ion spray LC-MS-MS.

Analyzing hair for many substances can be tedious and expensive, and a rapid screening method should prove helpful. Generally, screening has been performed using immunological tests, mainly in workplace drug testing, where the number of samples has been high. The aim of this study was to develop an LC-MS-MS method for the simultaneous analysis of several drugs of abuse in human hair as an alternative to immunological screening tests. In 75 randomly selected autopsy cases, hair was analyzed in addition to the usual specimens of blood and urine. The method included nicotine, cotinine, morphine, codeine, 6-acetylmorphine, ethylmorphine, amphetamine, methamphetamine, MDA, MDMA, benzoylecgonine, cocaine, 7-aminoflunitrazepam and diazepam. The LC-MS-MS analysis was performed on a SCIEX API 2000 MS-MS instrument equipped with an electrospray interface. To 20-50 mg of hair, 0.5 ml of mobile phase A (acetonitril:methanol:20 mM formate buffer, pH 3.0 (10:10:80)) and 25 microl of internal standard were added and the sample was incubated in a water bath at 37 degrees C during 18 h. Using a threshold of 20 ng/sample, equivalent to 1 ng/mg if 20mg hair is used, 26 positive results were found in 16 cases. Three of the 26 positive detections could not be confirmed by GC-MS. Two of the cases were not previously known as drug users. Of the 59 negative cases, only one case had a positive blood sample showing 0.01 and 0.07 microg/g femoral blood of 6-acetylmorphine and morphine, respectively. This might indicate drug abstinence resulting in decreased tolerance or even a "first time" use of heroin resulting in death. We conclude that the use of hair analysis in postmortem cases can reveal both unknown drug use, as well as confirm a period of drug abstinence prior to an acute fatal overdose. The proposed LC-MS-MS method showed high sensitivity, was very easy to perform and seemed appropriate for screening purposes.