Abstract The relaxin-3/relaxin family peptide receptor 3 (RXFP3) neuropeptidergic system is emerging as a potential target for treating various neuropsychiatric diseases, particularly those involving dysregulated stress and arousal. RXFP3 is abundantly expressed in several hypothalamic nuclei, and in the zona incerta (ZI). These regions play a central role in the regulation of stress and arousal, however the function of relaxin-3/RXFP3 within these circuits is unknown. The purpose of this study was to begin characterising this function by describing the distribution and genetic signature of neurons that express RXFP3. We used RNAscope fluorescent in situ hybridisation to characterise the spatial expression pattern and neurochemical phenotype of cells expressing Rxfp3 mRNA throughout the mouse lateral hypothalamus (LH) and ZI. We found that Rxfp3 is expressed across the rostrocaudal extent of both the LH and ZI and follows a parabolic pattern of expression, peaking in more rostral areas of each nucleus. Neurochemical phenotyping of Rxfp3 + cells with Gad1 , Slc17a6 (vGlut2), Pvalb , Th , and Sst showed that LH/ZI Rxfp3 + cells co-express each marker to varying extents, generally proportional to their overall abundance within each structure. Furthermore, LH/ZI Rxfp3+ cells overlapped with several known populations involved in various facets of fear learning and defensive behaviour, such as the dopaminergic A13 group, somatostatin-expressing rostral ZI neurons, and glutamatergic LH neurons. The neurochemical diversity of these neurons may reflect the overall role of both the LH and ZI as global regulators of behaviour and the role of relaxin-3/RXFP3 signalling in modulating high-vigilance states.

AIMS:Chronic methamphetamine (METH) intake is associated with a high risk of psychosis and high susceptibility to relapse following abstinence. Prelimbic cortex (PLC) extrasynaptic GABAA receptors containing the δ-subunit may be involved in the long-term effects of METH. We therefore investigated the effects of intra-PLC injection of the δ-subunit GABAA receptor agonist 4,5,6,7-tetrahydroisoxazolo (5,4-c)pyridine-3(-ol) (THIP), on METH sensitisation, a model of METH psychosis, or relapse to METH seeking behaviour following extinction of intravenous self-administration. METHODS AND RESULTS:In Experiment 1, male rats received 1 week of chronic treatment with METH (1 mg/kg days 1 and 7, 5 mg/kg days 2-6) or saline, followed by 14 days withdrawal. THIP (0, 0.1, 0.3 or 1.0 μg per hemisphere) was then micro-injected into the PLC prior to challenge with METH (1 mg/kg) or saline. Acute METH challenge enhanced locomotor hyperactivity in METH-pretreated rats, confirming sensitisation. THIP administration into the PLC dose-dependently reduced the effect of acute METH challenge in METH-pretreated rats. In Experiment 2, rats were trained to intravenously self-administer METH, followed by 2 weeks of extinction. Rats were then given a METH priming injection or exposed to cues associated with METH self-administration, both of which reinstated responding on the lever previously paired with METH delivery. At the 0.3 µg dose, injection of THIP into the PLC reduced reinstatement responding in both paradigms. CONCLUSION:Selective activation of extrasynaptic PLC δ-subunit GABAA receptors reduced METH sensitisation and drug primed or cue-induced METH-seeking behaviour. These findings may aid development of effective pharmacotherapies for treating chronic METH use.

In preclinical models, the non-intoxicating cannabis component cannabidiol (CBD) reduces relapse to methamphetamine (Meth)-seeking and Meth-induced hyperactivity in rats. Cannabis products containing multiple cannabinoids ("full spectrum") may offer greater therapeutic potential than single cannabinoid ("isolate") products. However, few studies tested this. This study examined whether a hemp extract (HE) containing multiple cannabinoids might be superior to CBD alone in reducing Meth-induced behavioural sensitisation and relapse, and whether serotonin 1 A receptors (5-HT1A) are involved. Male Sprague-Dawley rats self-administered either Meth or sucrose via lever press, followed by extinction and reinstatement by Meth injection (1 mg/kg; i.p.) or sucrose access. Rats received vehicle, CBD isolate (80 mg/kg), HE (containing 2.5 mg/kg of CBD and other phytocannabinoids), or HE with CBD added to match the 80 mg/kg amount of the CBD isolate (CBD + HE condition). The 5-HT1A antagonist WAY-100635 was co-administered to assess receptor involvement. Separate rats were tested for conditioned place preference (CPP) to assess possible intrinsic rewarding properties of the cannabinoids. A final group was tested for Meth-induced behavioural sensitisation. All CBD containing treatments reduced Meth-primed reinstatement, with HE and CBD + HE more effective than CBD isolate. There was no effect of any treatment on reacquisition of sucrose seeking. WAY-100635 did not block the effects of any treatment. Neither cannabinoid treatment produced CPP. All treatments reduced the expression of Meth-induced sensitised hyperactivity with CBD + HE showing some superiority over CBD or HE alone. This study suggests that CBD + HE may be more effective than CBD in reducing Meth relapse-like behaviour.

Abstract Craving – the powerful urge to seek and consume alcohol in response to alcohol-associated cues does not diminish after drinking cessation but rather is magnified throughout abstinence. This phenomenon, termed “incubation of craving”, contributes to the relapsing nature of alcohol use disorder. Despite its occurrence in human populations and being well-studied in rodent models of psychostimulant drug relapse, the underlying neural mechanisms and potential treatments remain largely unexplored for alcohol-related incubation of craving. Our research seeks to meet this gap, and this particular study investigated the neural correlates of the incubation of craving for alcohol-associated cues and assessed whether exercise could prevent increased relapse propensity in rats. Male Long Evans rats were trained to lever press for an alcohol reward delivered with simultaneous presentation of a discrete cue. This response was then extinguished and reinstated by presenting the discrete cue alone when rats pressed the lever. Cue-induced reinstatement occurred either on day 1 following extinction (No Abstinence) or on day 29 (Abstinence). A third group was tested on day 29 and had 4-hour daily voluntary running wheel access throughout this abstinence period (Exercise). All rats were perfused 90 minutes following test, and relative activation across the brain was estimated by quantifying c-Fos protein immunoreactivity. The brain-wide coordination of neural activity was also mapped. We found a robust incubation of craving effect for alcohol-associated cues, which was mitigated by exercise. Immunohistochemistry revealed that the Abstinence group demonstrated higher c-Fos immunoreactivity compared to the No Abstinence group in multiple reinstatement-related brain regions. This effect was reversed in the Exercise group. Brain-wide neural mapping demonstrated that the Abstinence group had decreased modularity (groups of coordinating brain regions) compared to the no-abstinence group. Although network connectivity profile in the exercise group was different from no abstinence, we found that overall neural activation returned to a similar modularity profile of clustered regions as this condition, indicating that exercise does not attenuate the incubation of craving effect by reversing all the neural effects of abstinence. Rather, exercise may be acting upon select brain regions or pathways to exert relapse protective effects by restoring widespread interconnectivity. This is the first study to investigate neural activation in incubated alcohol-seeking, and provides supporting evidence for promoting voluntary exercise as an adjunctive treatment for relapse prevention in alcohol-use disorder.

Relaxin family peptide receptor-3 (RXFP3) is a ligand-activated G-protein coupled receptor and the cognate receptor for the conserved neuropeptide relaxin-3. We recently demonstrated that chemogenetically activating an RXFP3-expressing population in the lateral hypothalamus and zona incerta (LH/ZI RXFP3 ) induced escape-like jumping behaviour following fear conditioning, but only in a subset of mice. Given the diverse hodology of the LH and ZI, we hypothesised that LH/ZI RXFP3 cells may consist of discrete subpopulations with unique connectivity patterns that govern different aspects of defensive behaviour. To explore this possibility, we unilaterally injected small volumes of a Cre-dependent anterograde viral tracer into four distinct sites of the LH/ZI in RXFP3-Cre mice and analysed their brain-wide efferent connectivity patterns. Each injection site group produced unique projection patterns, particularly to nuclei involved in threat and defensive behaviour. Of note were strong projections from the rostral ZI and anterior LH to the lateral habenula, and projections from the intermediate and caudal ZI to the ventrolateral periaqueductal gray. By combining retrograde tracing and RNAscope fluorescent in situ hybridisation, we identified that most LH/ZI RXFP3 projections to the lateral habenula arose from a subset of vGlut2-expressing lateral hypothalamus neurons, while most projections to the ventrolateral periaqueductal gray arose from a subset of GAD1-expressing zona incerta neurons. Taken together, our results strongly suggest that LH/ZI RXFP3 cells exhibit distinct efferent projection patterns throughout the brain depending on their topographical location within these nuclei, likely reflecting the functional diversity of these neurons.

Alcohol use disorder (AUD) is a complex neurological disorder with limited treatments available. Thus, understanding the neurobiological changes associated with AUD is crucial for the development of effective therapeutic approaches. Analysis of human post-mortem tissue provides insight into the long-term effects of alcohol use and cellular changes that contribute to addiction behavior. Here, we provide a collection of cellular changes found in human AUD post-mortem brain tissue, revealing brain region-specific cellular adaptations that map a complex neurobiological landscape of addiction. Examination across the cortex, striatum, hippocampus, hypothalamus, cerebellum, and midbrain reveals that although degeneration, metabolic disruption, and neuroinflammatory processes are common cellular processes that are impacted, these changes are distinctive by brain region and cell type. For example, white matter loss dominates in the prefrontal cortex, the hippocampus is sensitive to glial cell loss, subtypes of hypothalamic neurons are disproportionately affected, and the striatum shows subregional changes. Although these cellular adaptations are brain region and cell type specific, this review of studies over the last 30 years suggests that neuropathology in AUD undergoes neural network reorganization. Whether these changes are a response to chronic alcohol use or underlying alcohol-seeking behavior is a limitation of post-mortem analysis. However, mechanistic studies of rodent models and convergent evidence from human post-mortem tissue have identified therapeutic candidates such as oxytocin and GLP-1. Insights from animal studies have also highlighted underexplored yet potentially pivotal regions in AUD, such as the anterior insular cortex and the ventral pallidum, in human studies. Other limitations include a lack of sex-specific analyses, the incorporation of advanced neuroscience tools, and multiple regional analyses. Integrating AUD human post-mortem tissue bridges preclinical and clinical research, providing an invaluable understanding of neural mechanisms underpinning AUD and potential avenues for targeted interventions.

CASE HISTORY Bailey was 10-year-old girl transferred to a tertiary care children's hospital in New South Wales, Australia, with a provisional diagnosis of functional seizures, a subtype of functional neurological disorder (FND), for assessment and treatment. On presentation, Bailey's episodes were characterized by increased respiratory rate (hyperventilation), whole-body shaking, a wide-eyed fear expression, and brief periods of "zoning out." Within a week following admission, the episodes lengthened significantly and were usually accompanied by confusion, an altered level of consciousness, and motor activation with extreme levels of arousal. Following repeat medical investigations, the working diagnosis of FND was changed to antibody negative inflammatory encephalitis comorbid with a neuropsychiatric syndrome. Background Bailey lived with her biological parents and younger brother. They identified as a close-knit family. She was conceived via in vitro fertilization (IVF). The family reported that the pregnancy, though uncomplicated, was moderately stressful due to the IVF process and its uncertainties. Bailey met all developmental milestones on time. Outside of some reported sleep difficulties in the first three years of life, no major adjustment or separation anxiety issues were reported. Bailey flourished in the early years of primary school. In Grades 4 and 5, however, she experienced difficulties with friendships and developed anxiety around school because her teacher, a new graduate, was overwhelmed (e.g., the teacher would cry in front of the class). Bailey became hesitant about walking to school independently, and worried about occurrences at school (e.g., her brother getting injured on the playground, her friends quarreling, or her teacher being upset). Bailey's parents reported that she was always a compassionate and caring young person. Medically, Bailey had always been well with no history of physical illness. Severe anxiety was reported on the maternal side (never treated with psychopharmacology) and heart and kidney disease were reported on the paternal side. History of Presenting Illness In November 2020, Bailey had a fever and persistent cough and was subsequently diagnosed, via a throat swab, with mycoplasma infection. Her mother and brother had similar symptoms. All three were treated with a course of azithromycin. The following month, Bailey experienced recurring headaches that were not relieved by simple analgesics (paracetamol and ibuprofen). She also started experiencing sleep disturbances, characterized by waking one hour after sleep onset in a fright and saying, for example, "I haven't been sleeping," "I never can sleep," and "I have been up." Her mother showed her videos as evidence that she had slept. During this time, Bailey also experienced nightmares about her parents being harmed. These caused her significant distress on waking. The sleep disturbances persisted for four to five weeks. Bailey also started displaying heightened and distressed behavior that was out of character and disproportionate to the situations. For example, when her pet fish, which she owned for two days, died, she cried for hours, screaming and calling all her relatives. In February 2021, Bailey fractured her toe and started using crutches as she was not able to bear weight. Bailey catastrophized the severity and prognosis of the toe injury. She reported beliefs that she might eventually need a wheelchair and end up a quadriplegic. Later that month, Bailey hit her foot at school and began feeling unwell with a very low-grade temperature. Once home, she took a shower and fell to the ground. She started to scream, "I cannot breathe," "I cannot see." She was tremulous and clammy, and remained slumped on the ground. When her parents tried to calm her, she was unresponsive. The event lasted for 50 minutes. Paramedics were called, and Bailey was taken to her local emergency department, where she was diagnosed as experiencing a panic attack. Five days later (March 2021), Bailey re-presented to our tertiary care hospital because she was experiencing ongoing panic attacks four to five times a day, lasting approximately 10 minutes each. She was admitted for three days of investigation, which returned a normal electroencephalogram (EEG) and blood panel. She was discharged with a diagnosis of panic attacks and a referral to the local Child and Adolescent Mental Health Services. Ten days later, Bailey presented to her local hospital again because her panic attacks were becoming longer and had changed in character. She was now experiencing 10–15 episodes a day, each lasting 10–50 minutes and sometimes culminating in whole-body shaking. Investigations included magnetic resonance imaging (MRI), repeat EEG, serum N-methyl-D-aspartate (NMDA) autoantibodies, lumbar puncture (LP), and cell, protein, and encephalitis panels, all of which were normal. Trials involving propranolol (0.5 mg, BD), clonidine (25 mcg, BD), and quetiapine (6.25 mg, as required every six hours) showed no symptom improvement (See Table 1).1-3 Bailey was given a working diagnosis of FND presenting as functional seizures, on a backdrop of panic attacks. Because Bailey's presentation was so extreme, she was transferred to a pediatrician at our tertiary care hospital.Table 1:: Psychopharmacology used throughout treatmentFollowing admission, a medical review confirmed that all necessary investigations had been performed. A neurology review confirmed the working diagnosis of functional seizures from clinical history, review of video material, and review of EEG data. Bailey and her family were referred to the Department of Psychological Medicine for admission assessment into the Mind-Body Program for FND.4,5 The family assessment took place via telehealth (due to the COVID-19 pandemic). Bailey's history of cumulative stress, with both psychological (associated with school) and physical (mycoplasma infection and fractured toe) stressors, coupled with panic attacks that had, with time, morphed into functional seizures, was consistent with FND. Bailey's Progress in the Mind-Body Program The Mind-Body Program is a rehabilitation setting for children disabled by FND.5 On a daily basis, it includes individual therapy to learn regulation skills for managing functional seizures,4 physiotherapy to build physical resilience and stress resistance,6,7 and school attendance. There are also weekly family sessions. For children who are very aroused, pharmacotherapy is initiated to help decrease arousal, thereby enabling children to participate in all components of the program. In such cases, medications are slowly withdrawn when children's arousal levels decrease through participation in the program. Over the course of Bailey's first few days, it became apparent to the team that, despite the lengthy medical workup, the diagnosis of FND was likely incorrect. Key indicators included the following: First, Bailey showed no therapeutic response to the low-dose medications—clonidine (up to 50 mcg, QID), quetiapine (up to 50 mg, TDS), and propranolol (initially 5 mg, TDS)—that were trialed to help downregulate her arousal. Only propranolol, when increased to a higher dose of 7.5 mg TDS, showed some effect; it helped contain Bailey's heart rate increases (which had been up to 140 beats/minute). Second, Bailey's functional seizure episodes became prolonged, lasting several hours at a time, and were marked by changes in level of consciousness, with only brief periods (typically one to five minutes) of orientation to time, place, and person. Usually, Bailey was unable to identify her parents and would ask nursing staff to "call an ambulance" despite being in the hospital. Third, the episodes took on a repetitive pattern: whole-body shaking, changes in level of consciousness (being unable to respond to external stimuli or soothing), and activation of the flight-or-fight response, involving aggression (yelling, screaming, and hitting) and attempts to abscond. The flight-or-fight response was coupled with continuous verbalized, fear-orientated cognitions, repeated over and over, pertaining to being kidnapped, taken away, and so on. And fourth, Bailey now displayed extreme sensitivity to light and sound. Bailey cycled through these various altered states throughout the day. The only time that she settled was at night, with no sleep disturbances reported or documented. Outside of an elevated heart rate during periods of acute distress, all of Bailey's other clinical observations (body temperature and blood pressure) fell within normal parameters. The team's hypothesis was that some neuroinflammatory process that was not determined in the medical workup may underpin her symptoms. Setting Up a Neurology-Psychiatry Team to Deliver an Integrated Neurobio-Psycho-Social Intervention It was clear that Bailey's presentation lay at the interface of neurology and psychiatry. In this context, the mind-body team combined forces with the neurology team run by RD to create a joint team that could deliver an integrated neurobio-psycho-social approach and bridge the neurology/psychiatry divide.8 Bailey's parents, who were very stressed by the deterioration of her health, found this approach comforting. They had previously presented to the hospital many times, and seen many different doctors. They were distressed that the medical system presented so few options for assessing and treating their daughter. The joint effort began with repeat medical investigations, including a brain MRI, EEG, computed tomography (CT) scan of the chest, abdomen, and pelvis (looking for neural chest tumors from the sympathetic-adrenal lineage secreting hormones or neuroactive chemicals), and LP (looking for autoantibodies, such as NMDA-receptor antibodies, that are part of a diagnostic panel for encephalitis). All investigations were normal except for elevated neopterin (102 [normal range 6–30 nmol/L]) in the cerebrospinal fluid (CSF). Neopterin is an organic compound, called a pteridine, produced by activated macrophages and dendritic cells in response to cellular immune activation.9,10 It has been identified as a useful biomarker to indicate acute neuroinflammation, particularly in disorders that lack specific biomarkers. CSF neopterin has been shown to have a high level of sensitivity for defining neuroinflammation, including encephalitis.10 In Bailey's case, the elevated neopterin was consistent with a neuroinflammatory condition (encephalitis) potentially amenable to immunomodulatory treatment. The revised diagnosis of antibody negative inflammatory encephalitis (suspected post-mycoplasma) accompanied by a neuropsychiatric syndrome was discussed with Bailey's parents.11 The neurology-psychiatry team also revised the working formulation. It was hypothesized that the mycoplasma infection in November 2020 had triggered an immune-inflammatory response that first manifested in chronic headaches; subsequently, in the emergence of panic attacks, distress, fear-related cognitions, nightmares, and functional seizures; and finally, in Bailey's current presentation consistent with encephalitis (the repeating pattern of episodes described above). The Neurobiological Inpatient Intervention In April 2021, Bailey was started on immunosuppressive therapies, including intravenous (IV) immunoglobulin (IVIG) (1 g/1 kg) for three days, followed by pulse IV methylprednisolone for five days, followed by rituximab 1000 mg. This intervention resulted in a small improvement to her symptoms, with a modest reduction in agitation levels and episode duration. A second dose of rituximab 1000 mg was administered 14 days later. Once again, there was only modest improvement in symptoms, with Bailey experiencing more awake periods and a slight decrease in duration and intensity of episodes. In the context of slow improvements, brain MRI and LP were repeated under anesthetic in May 2021, returning normal MRI and CSF results, including neopterin. Ketamine was administered as a pre-procedural sedative (200 mg liquid oral). CH attended the procedure as a support person to help manage any behavioral escalations. CH noticed that following administration of ketamine, Bailey immediately became more orientated to the environment. She was able to communicate in full sentences and move around her bed. She was also able to recall recently presented information (e.g., the names of the doctors performing the procedures), direct nursing staff (asking for a cannula to be inserted in a certain location), and make requests (asking for certain objects around the room), all of which Bailey was unable to do prior to ketamine administration. She displayed no sensitivity to light and sound, which had been very prominent throughout her clinical course. Following anesthesic ketamine administration, Bailey returned to her baseline state of altered consciousness and patterns of repetitive episodes. Based on her clinical improvement on ketamine, the neurology-psychiatry team hypothesized that, even though NMDA antibodies had not been found with LP, possible glutamatergic process (given the mechanism of action of ketamine on the NMDA receptor) was potentially involved in some way. Based on this working hypothesis, amantadine, which also works on the NMDA receptor pathway, was identified as a potentially useful medication. Amantadine has a related mechanism on glutamate inhibition secondary to NMDA receptor antagonism and has been used in treatment-resistant neuropsychiatric syndromes.12 Approval was received through the therapeutic goods Special Access Scheme to trial amantadine with a starting dose of 25 mg twice daily. Bailey received her first dose of Amantadine on May 28, 2021. Following administration, Bailey's symptoms improved rapidly over a three-day period. Initially, she became more aware of her environment. Then she began responding appropriately to environmental stimuli (e.g., laughing when a family member made a joke). On the third day, she regained capacity to communicate on waking (e.g., "Good morning, Mummy"). A week after amantadine initiation, Bailey continued improving, however, she still displayed transient long-term memory recall difficulties (e.g., what school is and the names of her friends). She also displayed some trouble with fine motor movement and experienced pain in her ankles when she attempted to bear weight. She retained no memory of the acute period of her illness (approximately three months). At this point, the neurology-psychiatry team implemented a rehabilitation approach. A speech therapist and occupational therapist worked alongside Bailey's physiotherapist to increase her capacities. Bailey slowly regained her gross and fine motor functioning and daily life activities. Substantial improvements in cognition were also observed across a one-week period; Bailey was able to recall memories prior to the illness (i.e., friends' names, which school she went to, etc.). Despite these improvements, Bailey showed significant anxiety when separated from her mother. This was not present prior to the illness. As such, the psychiatry team, along with Bailey's parents, developed a graded separation-exposure plan to assist Bailey in managing her anxiety. Two weeks following initiation of amantadine, Bailey deteriorated and started experiencing a sharp headache in the forehead region above her left eye, increased irritability, and brief periods of severe distress characterized by high-pitched noises and amotivation to engage in rehabilitation. Bailey's parents and the treatment team struggled to contain their anxiety over the potential relapse. Additional IVIG and pulse IV methylprednisolone (three days) were administered. Bailey responded well to the treatment, with decreased headaches, reduction in irritability, and resolution of severe distress. Following this intervention, Bailey was cleared by all medical and allied health teams and was discharged home in July 2021, 110 days after her transfer to our hospital and 26 days after starting amantadine. Bailey's parents were asked to email a weekly update to the neurology clinical nurse consultant. A weekly telehealth follow-up with the mind-body team—a short review with Bailey (with her parents present) followed by a parent session—was also organized to support rehabilitation in the home setting. Relapse, Readmission, and the Second Attempt at Discharge After discharge, Bailey's recovery was variable. Initially, she had a recurrence of severe headaches and acute periods of distress, albeit less severe. She also experienced prolonged panic attacks (four to five times a day). During a short readmission, an additional round of IVIG and pulse IV methylprednisolone (three days) was completed, with minimal symptom relief. Medical investigations were rerun, including an MRI and LP. Throughout the investigations and the period waiting for results, the joint psychiatry-neurology team spent considerable time supporting and managing the family's distress regarding Bailey's deterioration. All investigations, including neopterin (10.83 nmol/L), returned normal results. Together, these findings suggested that there was no longer a neuroinflammatory driver for Bailey's decline. During the short readmission, the mind-body team and Bailey's family investigated running a formal inpatient rehabilitation program to increase Bailey's psychological and physical resilience and functioning. It soon became clear, however, that Bailey was unable to manage the separation from her parents required to participate. Among other behaviors, she exhibited prolonged panic attacks, lasting up to five hours, when separated to attend hospital school. During Bailey's initial admission, when functional seizures were the working diagnosis, attending the hospital school had been very distressing for both Bailey and her parents. Although she had limited recall from that time, both her parents and the team considered it likely that traumatic memories may have triggered both Bailey and her parents, undercutting her capacity to engage in this central element of the inpatient rehabilitation program. After considerable reflection, Bailey's parents and the team came to a joint agreement that the next rehabilitation steps would need to be undertaken in a slow and protracted manner, and at home. Because the panic attacks were so extreme—Bailey was unable to leave the house to attend outpatient physiotherapy— Bailey was started on a lithium trial (titrating up to 250 mg BD, with a blood level just under 0.5 mEq/L) to see if its neurostabilizing and neuroprotectant actions would help settle her neural networks and central nervous system (CNS). Lithium can reduce excitatory glutamate neurotransmission and increase inhibitory (gamma-aminobutyric acid, GABA) neurotransmission, overall reducing or "dampening down" excitatory neurotransmission in the brain.13 Further, lithium has been identified as a potential therapeutic strategy from treatment of neuropsychiatric conditions due to its ability to modulate autophagy.14 It was hypothesized that lithium may hold utility in Bailey's case through these aforementioned mechanisms. Our clinical impression was that lithium treatment had the desired effect, setting the scene for a home-based rehabilitation program. Rehabilitation in the Home Setting Over a six-month period, Bailey engaged in home rehabilitation with the support of her parents. Within this program, and through telehealth processes, Bailey engaged in weekly physiotherapy (with a community physiotherapist) and weekly psychology sessions (with a mind-body team member) focusing on cognitive behavioral therapy for anxiety and trauma-focused cognitive behavioral therapy. Likewise, Bailey's parents attended weekly and then biweekly parent sessions. Bailey also participated in daily exposure therapy implemented by her parents. Due to the severity of Bailey's illness, her parents experienced significant distress and caregiver burden during the rehabilitation process. The family bore much of the emotional demands of looking after an unwell child with an unknown illness trajectory. They also implemented a formal home rehabilitation program and negotiated with the medical system to establish a treatment team based in the community. Not surprisingly, Bailey's parents were hyperalert to infectious symptoms of any kind (e.g., sore throat, cough, headache) and to their daughter's repeated vocalizations about headache or fear. All of these symptoms were trauma triggers for the family. The team met with the family weekly (via telehealth) for support sessions as they struggled with these challenges and progressed through home rehabilitation. Over a period of 12 months, Bailey responded exceptionally well to the home rehabilitation program. She was able to return to school full time and transition to high school (in Australia) at the start of 2022. The Third Relapse In July 2022, Bailey injured her knee while running in her backyard. Soon after, Bailey's mother spent a night away from home. Additionally, her father contracted COVID-19 while on an overseas work trip. When he returned, the rest of the family also contracted COVID-19. These events occurred just prior to Bailey's return to school after a two-week break. Bailey previously verbalized worries about returning to school. And now, as the start of school approached, Bailey experienced symptom relapse. She had prolonged panic attacks characterized by rocking on her side, frothing at the mouth, and becoming unresponsive. Additionally, she experienced memory loss (including an inability to recognize her father), loss of the ability to speak and read, and incontinence of her bowels and bladder. Bailey was readmitted to our hospital in October 2022 for a repeat brain MRI, LP, and blood tests. All results were in the normal range, with no neuroinflammation process identified. Bailey's constellation of symptoms was consistent with a functional presentation, especially given that the symptoms emerged in the context of cumulative stress. The neurology-psychiatry team initially reviewed the new formulation with Bailey's parents, and then with Bailey. The team used a visual representation to depict the mycoplasma infection triggering two different processes (see Figure 1), an inflammatory process that underpinned the encephalitis (now resolved) and a stress-system activation process that underpinned the panic attacks and functional somatic symptoms (ongoing).Figure 1: This figure was used with Bailey and her family to depict the two processes activated by the mycoplasma infection. The first, an inflammatory process, was responsible for some of Bailey's symptoms during the first stage of illness (when she was becoming unwell) and for Bailey's symptoms during the second stage of illness (during her hospital admission). The second process, activation of the stress system, was responsible for most of Bailey's symptoms during the first stage of illness (when she was becoming unwell), for some of Bailey's symptoms during the second stage of illness (during her hospital admission), and for all of Bailey's symptoms during the third stage of illness (rehabilitation post hospital admission).Building on this formulation, the neurology-psychiatry team suggested that Bailey engage in a treatment program for her panic attacks and functional symptoms, just like any other child presenting with these symptoms. Because of Bailey's ongoing high arousal levels, the team suggested that her community psychiatrist consider trialing guanfacine as part of the transition process. It was anticipated that guanfacine could help downregulate Bailey's arousal, enabling her to engage in the therapeutic interventions (physiotherapy and psychotherapy) needed to address her functional symptoms. The team also encouraged the family to reinstate the home rehabilitation program. Transition to Community Care Following this admission, in October 2022 Bailey and her family were transitioned to a community treatment team (including psychologist, child and adolescent psychiatrist [who saw the family as a unit], and pediatrician). They provided ongoing support in implementing the mind-body and family interventions to treat Bailey's anxiety and functional symptoms. With this help, Bailey's symptoms began to improve. In November 2022, now at the age of 12, her memory impairments, including her inability to recognize her father, resolved, much to the relief of her family. Her panic attacks also decreased to two short episodes daily. By December, Bailey began speaking again and regained her ability to read and write. She was also able to complete outings with her family with minimal distress and attend physiotherapy sessions independently. Just prior to Christmas, Bailey visited school several times as an exposure task and engaged with a tutor to prepare for reintegrating back into school in 2023. In December 2023, Bailey was doing incredibly well. Her transition was progressing, with part-time enrollment in mainstream school and discontinuation of lithium. For ongoing management of anxiety and arousal, she was maintained on fluvoxamine 125 mg/day, guanfacine 2 mg/day, and propranolol 10 mg TDS. Bailey no longer experienced functional seizures and her mood stabilized. She was engaging well with her family and continuing to improve her engagement with friends and others. QUESTIONS TO THE CONSULTANTS − How do we integrate neurobio-psycho-social interventions in the treatment of neuropsychiatrically complex syndromes? − What do we know regarding the use/importance of inflammatory markers in the diagnosis of autoimmune encephalitis? − What do we know regarding the pharmacological management of neuropsychiatric syndromes with comorbid functional neurological symptoms? David L. Perez MD, MMSc This diagnostically and therapeutically complex case is illustrative of the need to integrate psychiatric and neurologic perspectives for patient-centered care in many clinical scenarios. Psychiatry and neurology have shared origins, with distinguished early leaders learning and working together in the Salpêtrière hospice in the late nineteenth and early twentieth centuries. For a multiplicity of theoretical, institutional, and practical reasons, psychiatry and neurology took distinct paths in the mid-1900s. For example, Archives of Neurology and Psychiatry split into distinct journals in 1960 (now JAMA Psychiatry and JAMA Neurology).15 Having two medical specialties dedicated to the assessment and management of ailments originating from the same organ system is a unique paradigm. The brain does not separate into neurologic and psychiatric circuits, however, and structure-function relationships are intimately connected neurobiologically. Left prefrontal cortex disruptions are associated with increased risk for post-stroke depression.16,17 Structural salience network alterations (i.e., dorsal anterior cingulate, anterior insula, dorsal amygdala, periaqueductal gray) are implicated in the pathophysiology of neurologic (e.g., migraine headaches, frontotemporal dementia) and psychiatric (e.g., mood, anxiety, and psychotic spectrum disorders) conditions, and in disorders that intersect both disciplines (e.g., FND).18-22 Numerous calls to bridge the great divide between neurology and psychiatry have been made over the past three decades by Eric Kandel, Joseph Martin, Bruce Price, and others.23-25 Convergent methodologies and neuroscientific breakthroughs have helped pave the way. This case highlights two clinically vibrant areas that benefit from an integrated neurology-psychiatry perspective, autoimmune encephalitis (AE) and FND. While initially aided by the discovery of autoantibodies for NMDA receptors (as well as by the book and film Brain on Fire), over a dozen distinct antibodies are now associated with autoimmune limbic encephalitis.26,27 In this diagnosis, subacute escalation of traditionally conceptualized psychiatric symptoms (e.g., anxiety, paranoia) can be part of the symptom complex (coupled with atypical features, such as rapid progression of other mental status changes). Initial assessment of these symptoms can be comprehensively evaluated by incorporating elements of psychiatric and neurologic expertise, including the characterization of atypical symptom evolution distinct from idiopathic psychiatric presentations. Furthermore, the evaluation of suspected autoimmune limbic encephalitis includes the acquisition and interpretation of biomarker data, such as review of neuroimaging, electrophysiology, and cerebrospinal fluid data. Similarly, chief complaints for FND are physical, including the spectrum of functional seizures, abnormal movements, limb weakness, speech/voice difficulties, dizziness, and cognitive concerns.28 Diagnosis of FND leverages neurologic expertise to appreciate positive, rule-in examination signs and semiological features. The neuropsychiatric and psychosocial complexity of FND is great, however, and the use of diagnostic features in isolation does not readily translate to patient-centered care in many instances.29 Thus, how might we integrate neurobio-psycho-social interventions in treating neuropsychiatrically complex patients? Below are three critical elements for doing so (among other considerations beyond the scope of this article): (1) increased educational cross-training, (2) embedded neurology-psychiatry models of care, and (3) institutional/programmatic considerations. Education The Accreditation Council for Graduate Medical Education in the United States requires two months of neurology training during psychiatry residency and at least one month of psychiatry training during neurology residency. Such limited cross-training has left neurologists ill-equipped to consider the mental health and psychosocial concerns frequently associated with neurological conditions. Similarly, psy

Rationale Alcopop beverages are generally the first alcoholic beverage that young females drink which contain high levels of sugar and alcohol. The over-consumption of these drinks may encourage alcohol co-administration with methamphetamine (METH) impacting on drinking behaviour and glial function.Aims The aims of this study were to evaluate the effect of adolescent binge alcohol exposure on consumption level, anxiety-like behaviour, cross-sensitization with METH and on astrocyte expression in reward related brain regions.Methods Adolescent female Sprague-Dawley rats had daily 1-hour oral alcohol consumption of alcopop (ALCP; with sucrose) or ethanol-only (ETOH; without sucrose), transitioned from 5 to 15% (v/v) ethanol content for 34 days. Water and sucrose groups act as controls. During alcohol withdrawal, rats were tested for anxiety on the elevated plus maze (EPM) and locomotor activity following saline or METH (1 mg/kg i.p) treatment. Brains were then collected to assess astrocyte immunofluorescence for glial fibrillary acidic protein (GFAP) in reward-related brain regions.Results Rats pretreated with 5% ALCP consumed significantly more volume and ethanol intake when compared to 5% EtOH rats. Both ALCP and EtOH groups had a higher preference ratio for 5% than 15% alcohol solutions and ALCP rats had greater ethanol intake at 15% than EtOH rats. Alcohol withdrawal showed no significant differences between groups on anxiety, METH cross-sensitization effects or GFAP intensity in the regions studied.Conclusions Overall, the addition of sucrose to alcoholic solutions encouraged female rats to consume larger volumes and greater ethanol intake compared to ethanol-only solutions, yet did not have long lasting effects on behaviour and astrocytes.

Cannabis has been used historically for both medicinal and recreational purposes, with the most notable cannabinoids being cannabidiol (CBD) and tetrahydrocannabinol (THC). Although their therapeutic effects have been well studied and their recreational use is highly debated, the underlying mechanisms of their biological effects remain poorly defined. In this study, we use isobaric tag-based sample multiplexed proteome profiling to investigate protein abundance differences in the human neuroblastoma SH-SY5Y cell line treated with CBD and THC. We identified significantly regulated proteins by each treatment and performed a pathway classification and associated protein–protein interaction analysis. Our findings suggest that these treatments may lead to mitochondrial dysfunction and induce endoplasmic reticulum stress. These data can potentially be interrogated further to investigate the potential role of CBD and THC in various biological and disease contexts, providing a foundation for future studies.

Cognitive flexibility is a crucial ability in humans that can be affected by chronic methamphetamine (METH) addiction. The present study aimed to elucidate the mechanisms underlying cognitive impairment in mice chronically administered METH via an oral self-administration method. Further, the effect of melatonin treatment on recovery of METH-induced cognitive impairment was also investigated. Cognitive performance of the mice was assessed using an attentional set shift task (ASST), and possible underlying neurotoxic mechanisms were investigated by proteomic and western blot analysis of the prefrontal cortex (PFC). The results showed that mice-administered METH for 21 consecutive days exhibited poor cognitive performance compared to controls. Cognitive deficit in mice partly recovered after METH withdrawal. In addition, mice treated with melatonin during METH withdrawal showed a higher cognitive recovery than vehicle-treated METH withdrawal mice. Proteomic and western blot analysis revealed that METH self-administration increased neurotoxic markers, including disruption to the regulation of mitochondrial function, mitophagy, and decreased synaptic plasticity. Treatment with melatonin during withdrawal restored METH-induced mitochondria and synaptic impairments. These findings suggest that METH-induced neurotoxicity partly depends on mitochondrial dysfunction leading to autophagy-dependent cell death and that the recovery of neurological impairments may be enhanced by melatonin treatment during the withdrawal period.

Introduction Methamphetamine (METH, “ice”) is a potent and addictive psychostimulant. Abuse of METH perturbs neurotransmitter systems and induces neurotoxicity; however, the neurobiological mechanisms which underlie addiction to METH are not fully understood, limiting the efficacy of available treatments. Here we investigate METH-induced changes to neuronal nitric oxide synthase (nNOS), parvalbumin and calretinin-expressing GABAergic interneuron populations within the nucleus accumbens (NAc), prefrontal cortex (PFC) and orbitofrontal cortex (OFC). We hypothesise that dysfunction or loss of these GABAergic interneuron populations may disrupt the excitatory/inhibitory balance within the brain. Methods Male Long Evans rats ( N = 32) were trained to lever press for intravenous METH or received yoked saline infusions. Following 14 days of behavioural extinction, animals were given a non-contingent injection of saline or METH (1 mg/kg, IP) to examine drug-primed reinstatement to METH-seeking behaviours. Ninety minutes post-IP injection, animals were culled and brain sections were analysed for Fos, nNOS, parvalbumin and calretinin immunoreactivity in eight distinct subregions of the NAc, PFC and OFC. Results METH exposure differentially affected GABAergic populations, with METH self-administration increasing nNOS immunoreactivity at distinct locations in the prelimbic cortex and decreasing parvalbumin immunoreactivity in the NAc. METH self-administration triggered reduced calretinin immunoreactivity, whilst acute METH administration produced a significant increase in calretinin immunoreactivity. As expected, non-contingent METH-priming treatment increased Fos immunoreactivity in subregions of the NAc and PFC. Conclusion Here we report that METH exposure in this model may alter the function of GABAergic interneurons in more subtle ways, such as alterations in neuronal firing or synaptic connectivity.

The prefrontal cortex plays a critical role in regulating substance use disorder and is strongly implicated in inhibition of and relapse to drug-seeking. Within the prefrontal cortex, inhibitory interneurons make up tightly regulated microcircuits that moderate activity of pyramidal neurons and hence control output to downstream circuits including those important for addiction-related behaviours. These interneurons can be classed into subpopulations based on peptide expression, however the complexity of these subclasses is only beginning to be understood. In this review we focus on four peptides that have receptor expression on interneurons and pyramidal neurons in the PFC. All four peptides have well-known roles in mediating drug-seeking behaviours, however their function within the prefrontal cortex is less well defined. We will review evidence from preclinical animal models that cells expressing these receptors are recruited during addiction-related behaviours, and that direct pharmacological manipulation of these systems within the prefrontal cortex may lead to altered drug-seeking behaviours. In our view, a deeper understanding of activity of specific microcircuits within this region, and their effect on pyramidal neuronal output are critical for understanding and remediating specific behaviours relevant to substance use disorder. Uncovering function of cells populations that express these neuropeptides and their receptors may help to define these microcircuits, and hence to understand how and why these behaviours are altered following drug exposure.

Early life stress (ELS) is associated with perturbed neural development and augmented vulnerability to mental health disorders, including addiction. How ELS changes the brain to increase addiction risk is poorly understood, and there are no therapies which target this ELS-induced vulnerability. ELS disrupts the oxytocin system, which can modulate addiction susceptibility, suggesting that targeting the oxytocin system may be therapeutic in this ELS-addiction comorbidity. Therefore, we determined whether adolescent oxytocin treatment after ELS could: (1) reduce vulnerability to anxiety, social deficits, and methamphetamine-taking and reinstatement; and (2) restore hypothalamic oxytocin and corticotropin-releasing factor expressing neurons and peripheral oxytocin and corticosterone levels. Long Evans pups underwent maternal separation (MS) for either 15 min or 360 min on postnatal days (PND) 1–21. During adolescence (PNDs 28–42), rats received a daily injection of either oxytocin or saline. In Experiment 1, adult rats were assessed using the elevated plus-maze, social interaction procedure, and methamphetamine self-administration procedure, including extinction, and cue-, methamphetamine- and yohimbine-induced reinstatement. In Experiment 2, plasma for enzyme immunoassays and brain tissue for immunofluorescence were collected from adult rats after acute stress exposure. Adolescent oxytocin treatment ameliorated ELS-induced anxiety and reduced methamphetamine- and yohimbine-induced reinstatement in both sexes, and suppressed methamphetamine intake and facilitated extinction in males only. Additionally, adolescent oxytocin treatment after ELS restored oxytocin-immunoreactive cells and stress-induced oxytocin levels in males, and attenuated stress-induced corticosterone levels in both sexes. Adolescent oxytocin treatment reverses some of the ELS effects on later-life psychopathology and vulnerability to addiction.

Rationale Stress exposure during adolescence contributes to developing a methamphetamine (METH) use disorder. However, most of the studies investigating addiction-related behaviours include only male rodents, despite METH addiction rates being higher in females. Furthermore, animal studies investigating the effects of stress on methamphetamine addiction have used only basic self-administration models which may not be sensitive to the effects of stress. Objectives This project explored whether adolescent isolation stress exposure increases the incidence of four key addiction-related behaviours in female rats. Methods Thirty-two female rat pups were caged in groups of four or individually during adolescence from postnatal (PND) day 22, with the latter being re-socialised in groups of four on PND 43. In adulthood, rats were tested for addiction-like behaviours in a METH self-administration paradigm modelling motivation to take METH, persistence in drug-seeking behaviour when METH was not available, resistance to extinction, and propensity to reinstate after a period of withdrawal. Results Adolescent social isolation resulted in lower METH intake during acquisition; however, the paradigm modelling drug-seeking when the drug was unavailable engendered intermittent METH bingeing in all rats, abolishing the group differences in intake during this phase. Adolescent social isolation also accelerated extinction of non-reinforced lever pressing, and increased stress-primed reinstatement, compared to the group-housed rats. Conclusions Adolescent social isolation stress alters various methamphetamine addiction-like behaviours in female rats.

The ability to discriminate competing external stimuli and initiate contextually appropriate behaviours is a key brain function. Neurons in the deep superior colliculus (dSC) integrate multisensory inputs and activate descending projections to premotor pathways responsible for orienting, attention and defence, behaviours which involve adjustments to respiratory and cardiovascular parameters. However, the neural pathways that subserve the physiological components of orienting are poorly understood. We report that orienting responses to optogenetic dSC stimulation are accompanied by short-latency autonomic, respiratory and electroencephalographic effects in awake rats, closely mimicking those evoked by naturalistic alerting stimuli. Physiological responses were not accompanied by detectable aversion or fear, and persisted under urethane anaesthesia, indicating independence from emotional stress. Anterograde and trans-synaptic viral tracing identified a monosynaptic pathway that links the dSC to spinally projecting neurons in the medullary gigantocellular reticular nucleus (GiA), a key hub for the coordination of orienting and locomotor behaviours. In urethane-anaesthetized animals, sympathoexcitatory and cardiovascular, but not respiratory, responses to dSC stimulation were replicated by optogenetic stimulation of the dSC-GiA terminals, suggesting a likely role for this pathway in mediating the autonomic components of dSC-mediated responses. Similarly, extracellular recordings from putative GiA sympathetic premotor neurons confirmed short-latency excitatory inputs from the dSC. This pathway represents a likely substrate for autonomic components of orienting responses that are mediated by dSC neurons and suggests a mechanism through which physiological and motor components of orienting behaviours may be integrated without the involvement of higher centres that mediate affective components of defensive responses.

Four groups of rats received by intratracheal instillation (1) a lead chromate paint particulate suspension, (2) lead tetraoxide suspension, (3) lead acetate solution, or (4) saline. Lead-dosed animals received an equivalent dose of 1 mg lead/kg body weight. Distribution of lead was monitored through assays of urine, feces, and tissues (lung, bone, muscle kidney, liver) obtained at post-mortem 5 weeks after exposure. Delta-aminolevulinie acid dehydratase (ALA-D) activity was measured to determine the effect of lead on heme biosynthesis. The vast majority of the dosed lead in the paint matrix remained in the lung. In contrast, in the lead acetate-dosed animals, little remained in the lung, but significant elevations were found in bone and kidney. Blood ADA-D was significantly depressed in the lead acetate-treated animals, but was not significantly different from control animals in the animals dosed with lead paint or lead tetraoxide. These findings suggest that lead chromate in an alkyd resin paint matrix is poorly absorbed from the lung compared with lead acetate and lead tetraoxide.