Accelerated neurodegeneration of basal forebrain cholinergic neurons in HIV-1 gp120 transgenic mice: Critical role of the p75 neurotrophin receptor.

Human Immunodeficiency Virus-1 (HIV) infection of the brain induces HIV-associated neurocognitive disorders (HAND). The set of molecular events employed by HIV to drive cognitive impairments in people living with HIV are diverse and remain not completely understood. We have shown that the HIV envelope protein gp120 promotes loss of synapses and decreases performance on cognitive tasks through the p75 neurotrophin receptor (p75NTR). This receptor is abundant on cholinergic neurons of the basal forebrain and contributes to cognitive impairment in various neurological disorders. In this study, we examined cholinergic neurons of gp120 transgenic (gp120tg) mice for signs of degeneration. We observed that the number of choline acetyltransferase-expressing cells is decreased in old (12-14-month-old) gp120tg mice when compared to age matched wild type. In the same animals, we observed an increase in the levels of pro-nerve growth factor, a ligand of p75NTR, as well as a disruption of consolidation of extinction of conditioned fear, a behavior regulated by cholinergic neurons of the basal forebrain. Both biochemical and behavioral outcomes of gp120tg mice were rescued by the deletion of the p75NTR gene, strongly supporting the role that this receptor plays in the neurotoxic effects of gp120. These data indicate that future p75NTR-directed pharmacotherapies could provide an adjunct therapy against synaptic simplification caused by HIV.

Role of Basal Forebrain Neurons in Adrenomyeloneuropathy in Mice and Humans.

OBJECTIVE: X-linked adrenoleukodystrophy is caused by mutations in the peroxisomal half-transporter ABCD1. The most common manifestation is adrenomyeloneuropathy, a hereditary spastic paraplegia of adulthood. The present study set out to understand the role of neuronal ABCD1 in mice and humans with adrenomyeloneuropathy. METHODS: Neuronal expression of ABCD1 during development was assessed in mice and humans. ABCD1-deficient mice and human brain tissues were examined for corresponding pathology. Next, we silenced ABCD1 in cholinergic Sh-sy5y neurons to investigate its impact on neuronal function. Finally, we tested adeno-associated virus vector-mediated ABCD1 delivery to the brain in mice with adrenomyeloneuropathy. RESULTS: ABCD1 is highly expressed in neurons located in the periaqueductal gray matter, basal forebrain and hypothalamus. In ABCD1-deficient mice (Abcd1-/y), these structures showed mild accumulations of α-synuclein. Similarly, healthy human controls had high expression of ABCD1 in deep gray nuclei, whereas X-ALD patients showed increased levels of phosphorylated tau, gliosis, and complement activation in those same regions, albeit not to the degree seen in neurodegenerative tauopathies. Silencing ABCD1 in Sh-sy5y neurons impaired expression of functional proteins and decreased acetylcholine levels, similar to observations in plasma of Abcd1-/y mice. Notably, hind limb clasping in Abcd1-/y mice was corrected through transduction of ABCD1 in basal forebrain neurons following intracerebroventricular gene delivery. INTERPRETATION: Our study suggests that the basal forebrain-cortical cholinergic pathway may contribute to dysfunction in adrenomyeloneuropathy. Rescuing peroxisomal transport activity in basal forebrain neurons and supporting glial cells might represent a viable therapeutic strategy. ANN NEUROL 2024;95:442-458.

Enriched environment ameliorates fear memory impairments induced by sleep deprivation via inhibiting PIEZO1/calpain/autophagy signaling pathway in the basal forebrain.

AIMS: To verify the hypothesis that an enriched environment (EE) alleviates sleep deprivation-induced fear memory impairment by modulating the basal forebrain (BF) PIEZO1/calpain/autophagy pathway. METHODS: Eight-week-old male mice were housed in a closed, isolated environment (CE) or an EE, before 6-h total sleep deprivation. Changes in fear memory after sleep deprivation were observed using an inhibitory avoidance test. Alterations in BF PIEZO1/calpain/autophagy signaling were detected. The PIEZO1 agonist Yoda1 or inhibitor GsMTx4, the calpain inhibitor PD151746, and the autophagy inducer rapamycin or inhibitor 3-MA were injected into the bilateral BF to investigate the pathways involved in the memory-maintaining role of EE in sleep-deprived mice. RESULTS: Mice housed in EE performed better than CE mice in short- and long-term fear memory tests after sleep deprivation. Sleep deprivation resulted in increased PIEZO1 expression, full-length tropomyosin receptor kinase B (TrkB-FL) degradation, and autophagy, as reflected by increased LC3 II/I ratio, enhanced p62 degradation, increased TFEB expression and nuclear translocation, and decreased TFEB phosphorylation. These molecular changes were partially reversed by EE treatment. Microinjection of Yoda1 or rapamycin into the bilateral basal forebrain induced excessive autophagy and eliminated the cognition-protective effects of EE. Bilateral basal forebrain microinjection of GsMTx4, PD151746, or 3-MA mimicked the cognitive protective and autophagy inhibitory effects of EE in sleep-deprived mice. CONCLUSIONS: EE combats sleep deprivation-induced fear memory impairments by inhibiting the BF PIEZO1/calpain/autophagy pathway.

Structural and molecular cholinergic imaging markers of cognitive decline in Parkinson's disease.

Cognitive decline in Parkinson's disease is related to cholinergic system degeneration, which can be assessed in vivo using structural MRI markers of basal forebrain volume and PET measures of cortical cholinergic activity. In the present study we aimed to examine the interrelation between basal forebrain degeneration and PET-measured depletion of cortical acetylcholinesterase activity as well as their relative contribution to cognitive impairment in Parkinson's disease. This cross-sectional study included 143 Parkinson's disease participants without dementia and 52 healthy control participants who underwent structural MRI, PET scanning with 11C-methyl-4-piperidinyl propionate (PMP) as a measure of cortical acetylcholinesterase activity, and a detailed cognitive assessment. Based on the fifth percentile of the overall cortical PMP PET signal from the control group, people with Parkinson's disease were subdivided into a normo-cholinergic (n = 94) and a hypo-cholinergic group (n = 49). Volumes of functionally defined posterior and anterior basal forebrain subregions were extracted using an established automated MRI volumetry approach based on a stereotactic atlas of cholinergic basal forebrain nuclei. We used Bayesian t-tests to compare basal forebrain volumes between controls, and normo- and hypo-cholinergic Parkinson's participants after covarying out age, sex and years of education. Associations between the two cholinergic imaging measures were assessed across all people with Parkinson's disease using Bayesian correlations and their respective relations with performance in different cognitive domains were assessed with Bayesian ANCOVAs. As a specificity analysis, hippocampal volume was added to the analysis. We found evidence for a reduction of posterior basal forebrain volume in the hypo-cholinergic compared to both normo-cholinergic Parkinson's disease [Bayes factor against the null model (BF10) = 8.2] and control participants (BF10 = 6.0), while for the anterior basal forebrain the evidence was inconclusive (BF10 < 3). In continuous association analyses, posterior basal forebrain volume was significantly associated with cortical PMP PET signal in a temporo-posterior distribution. The combined models for the prediction of cognitive scores showed that both cholinergic markers (posterior basal forebrain volume and cortical PMP PET signal) were independently related to multi-domain cognitive deficits, and were more important predictors for all cognitive scores, including memory scores, than hippocampal volume. We conclude that degeneration of the posterior basal forebrain in Parkinson's disease is accompanied by functional cortical changes in acetylcholinesterase activity and that both PET and MRI cholinergic imaging markers are independently associated with multi-domain cognitive deficits in Parkinson's disease without dementia. Comparatively, hippocampal atrophy only seems to have minimal involvement in the development of early cognitive impairment in Parkinson's disease.

Distribution of Vasopressin 1a and Oxytocin Receptor Binding in the Basal Forebrain and Midbrain of Male and Female Mongolian Gerbils.

The nonapeptide system modulates a diversity of social behaviors, including aggression, parental care, affiliation, sexual behavior, and pair bonding. Such social behaviors are regulated through oxytocin and vasopressin activation of the oxytocin receptor (OXTR) and vasopressin V1a receptor (AVPR1A) in the brain. Nonapeptide receptor distributions have been mapped for several species, however, studies have demonstrated that there is substantial variation across species. Mongolian gerbils (Meriones unguiculatus) are an excellent organism for studying family dynamics, social development, pair bonding, and territorial aggression. Although an increasing number of studies are examining the neural mechanisms of social behavior in Mongolian gerbils, nonapeptide receptor distributions have yet to be characterized for this species. Here we conducted receptor autoradiography to map distributions of OXTR and AVPR1A binding throughout the basal forebrain and midbrain of female and male Mongolian gerbils. Further, we assessed whether gonadal sex influenced binding densities in brain regions important for social behavior and reward, however, we observed no effects of sex on OXTR or AVPR1A binding densities. These findings provide mapping distributions of nonapeptide receptors in male and female Mongolian gerbils, laying a foundation for future studies that seek to manipulate the nonapeptide system to examine nonapeptide-mediated social behavior.

Basal forebrain cholinergic neurons are vulnerable in a mouse model of Down syndrome and their molecular fingerprint is rescued by maternal choline supplementation.

Basal forebrain cholinergic neuron (BFCN) degeneration is a hallmark of Down syndrome (DS) and Alzheimer's disease (AD). Current therapeutics in these disorders have been unsuccessful in slowing disease progression, likely due to poorly understood complex pathological interactions and dysregulated pathways. The Ts65Dn trisomic mouse model recapitulates both cognitive and morphological deficits of DS and AD, including BFCN degeneration and has shown lifelong behavioral changes due to maternal choline supplementation (MCS). To test the impact of MCS on trisomic BFCNs, we performed laser capture microdissection to individually isolate choline acetyltransferase-immunopositive neurons in Ts65Dn and disomic littermates, in conjunction with MCS at the onset of BFCN degeneration. We utilized single population RNA sequencing (RNA-seq) to interrogate transcriptomic changes within medial septal nucleus (MSN) BFCNs. Leveraging multiple bioinformatic analysis programs on differentially expressed genes (DEGs) by genotype and diet, we identified key canonical pathways and altered physiological functions within Ts65Dn MSN BFCNs, which were attenuated by MCS in trisomic offspring, including the cholinergic, glutamatergic and GABAergic pathways. We linked differential gene expression bioinformatically to multiple neurological functions, including motor dysfunction/movement disorder, early onset neurological disease, ataxia and cognitive impairment via Ingenuity Pathway Analysis. DEGs within these identified pathways may underlie aberrant behavior in the DS mice, with MCS attenuating the underlying gene expression changes. We propose MCS ameliorates aberrant BFCN gene expression within the septohippocampal circuit of trisomic mice through normalization of principally the cholinergic, glutamatergic, and GABAergic signaling pathways, resulting in attenuation of underlying neurological disease functions.

Dopamine D1-receptor-expressing pathway from the nucleus accumbens to ventral pallidum-mediated sevoflurane anesthesia in mice.

BACKGROUND: General anesthesia has long been used in clinical practice, but its precise pharmacological effects on neural circuits are not fully understood. Recent investigations suggest that the sleep-wake system may play a role in the reversible loss of consciousness induced by general anesthetics. Studies in mice have shown that microinjection of dopamine receptor 1 (D1R) agonists into the nucleus accumbens (NAc) promotes recovery from isoflurane anesthesia, while microinjection of D1R antagonists has the opposite effect. Furthermore, during the induction and maintenance of sevoflurane anesthesia, there is a significant decrease in extracellular dopamine levels in the NAc, which subsequently increases during the recovery period. These findings suggest the involvement of the NAc in the regulation of general anesthesia. However, the specific role of D1R-expressing neurons in the NAc during general anesthesia and the downstream effect pathways are still not well understood. METHODS: In order to analyze the impact of sevoflurane anesthesia on NAcD1R neurons and the NAcD1R -VP pathway, this study employed calcium fiber photometry to investigate alterations in the fluorescence intensity of calcium signals in dopamine D1-receptor-expressing neurons located in the nucleus accumbens (NAcD1R neurons) and the NAcD1R -VP pathway during sevoflurane anesthesia. Subsequently, optogenetic techniques were utilized to activate or inhibit NAcD1R neurons and their synaptic terminals in the ventral pallidum (VP), aiming to elucidate the role of NAcD1R neurons and the NAcD1R -VP pathway in sevoflurane anesthesia. These experiments were supplemented with electroencephalogram (EEG) recordings and behavioral tests. Lastly, a genetically-encoded fluorescent sensor was employed to observe changes in extracellular GABA neurotransmitters in the VP during sevoflurane anesthesia. RESULTS: Our findings revealed that sevoflurane administration led to the inhibition of NAcD1R neuron population activity, as well as their connections within the ventral pallidum (VP). We also observed a reversible reduction in extracellular GABA levels in the VP during both the induction and emergence phases of sevoflurane anesthesia. Additionally, the optogenetic activation of NAcD1R neurons and their synaptic terminals in the VP resulted in a promotion of wakefulness during sevoflurane anesthesia, accompanied by a decrease in EEG slow wave activity and burst suppression rate. Conversely, the optogenetic inhibition of the NAcD1R -VP pathway exerted opposite effects. CONCLUSION: The NAcD1R -VP pathway serves as a crucial downstream pathway of NAcD1R neurons, playing a significant role in regulating arousal during sevoflurane anesthesia. Importantly, this pathway appears to be associated with the release of GABA neurotransmitters from VP cells.

Vesicular acetylcholine transporter in the basal forebrain improves cognitive impairment in chronic cerebral hypoperfusion rats by modulating theta oscillations in the hippocampus.

The cholinergic transmission in the medial septum and ventral limb of the diagonal band of broca (MS/VDB)-hippocampal circuit and its associated theta oscillations play a crucial role in chronic cerebral hypoperfusion (CCH)-related cognitive impairment. However, the contribution and mechanism of the vesicular acetylcholine transporter (VAChT), a vital protein that regulates acetylcholine (ACh) release, in CCH-related cognitive impairment are not well understood. To investigate this, we established a rat model of CCH by performing 2-vessel occlusion (2-VO) and overexpressed VAChT in the MS/VDB via stereotaxic injection of adeno-associated virus (AAV). We evaluated the cognitive function of the rats using the Morris Water Maze (MWM) and Novel Object Recognition Test (NOR). We employed enzyme-linked immunosorbent assay (ELISA), Western blot (WB), and immunohistochemistry (IHC) to assess hippocampal cholinergic levels. We also conducted in vivo local field potentials (LFPs) recording experiments to evaluate changes in hippocampal theta oscillations and synchrony. Our findings showed that VAChT overexpression shortened the escape latency in the hidden platform test, increased swimming time in the platform quadrant in probe trains, and increased the recognition index (RI) in NOR. Moreover, VAChT overexpression increased hippocampal cholinergic levels, improved theta oscillations, and improved the synchrony of theta oscillations between CA1 and CA3 in CCH rats. These results suggest that VAChT plays a protective role in CCH-induced cognitive deficits by regulating cholinergic transmission in the MS/VDB-hippocampal circuit and promoting hippocampal theta oscillations. Therefore, VAChT could be a promising therapeutic target for treating CCH-related cognitive impairments.

Antisense oligonucleotides targeting basal forebrain ATXN2 enhances spatial memory and ameliorates sleep deprivation-induced fear memory impairment in mice.

INTRODUCTION: Regulation of brain-derived neurotrophic factor (BDNF) in the basal forebrain ameliorates sleep deprivation-induced fear memory impairments in rodents. Antisense oligonucleotides (ASOs) targeting ATXN2 was a potential therapy for spinocerebellar ataxia, whose pathogenic mechanism associates with reduced BDNF expression. We tested the hypothesis that ASO7 targeting ATXN2 could affect BDNF levels in mouse basal forebrain and ameliorate sleep deprivation-induced fear memory impairments. METHODS: Adult male C57BL/6 mice were used to evaluate the effects of ASO7 targeting ATXN2 microinjected into the bilateral basal forebrain (1 µg, 0.5 µL, each side) on spatial memory, fear memory and sleep deprivation-induced fear memory impairments. Spatial memory and fear memory were detected by the Morris water maze and step-down inhibitory avoidance test, respectively. Immunohistochemistry, RT-PCR, and Western blot were used to evaluate the changes of levels of BDNF, ATXN2, and postsynaptic density 95 (PSD95) protein as well as ATXN2 mRNA. The morphological changes in neurons in the hippocampal CA1 region were detected by HE staining and Nissl staining. RESULTS: ASO7 targeting ATXN2 microinjected into the basal forebrain could suppress ATXN2 mRNA and protein expression for more than 1 month and enhance spatial memory but not fear memory in mice. BDNF mRNA and protein expression in basal forebrain and hippocampus was increased by ASO7. Moreover, PSD95 expression and synapse formation were increased in the hippocampus. Furthermore, ASO7 microinjected into the basal forebrain increased BDNF and PSD95 protein expression in the basal forebrain of sleep-deprived mice and counteracted sleep deprivation-induced fear memory impairments. CONCLUSION: ASOs targeting ATXN2 may provide effective interventions for sleep deprivation-induced cognitive impairments.

Histamine and its H 1 receptors in the ventral pallidum mediate formalin-induced pain-related behaviors through this region and spinal cord opioid receptors.

Many structures of the central nervous system recruit different neurotransmitters in pain processing. This study focused on the contribution of histamine and its H 1 receptors in the ventral pallidum (VP) in mediating pain-triggered behaviors. Intra-VP microinjection of histamine and 2-pyridylethylamine (2-PEA, a histamine H 1 receptor agonist) at the same doses of 0.5 and 1 µg/200 nl reduced both the first and second phases of licking/biting duration as well as flinching number induced by intra-plantar (ipl) injection of formalin (2.5%, 50 µl). Premicroinjection of mepyramine (a histamine H 1 antagonist, 2 µg/200 nl) into the VP antagonized the suppressive effects of 1 µg/200 nl histamine and 2-PEA on licking/biting and flinching behaviors. The possible mechanisms of the above-mentioned pain-reducing effects were followed by intra-VP and intrathecal administration of naloxone (an opioid receptor antagonist). Naloxone (2 µg/200 nl) preadministration into the VP inhibited attenuating effects of histamine and 2-PEA on both the licking/biting and flinching behaviors, whereas intrathecal injection of naloxone only inhibited their suppressing effects on flinching behavior. None of the treatments used in this study altered the animal's motor activity. The obtained results may reveal the role of histamine and its activated H 1 receptor in the VP in suppressing the pain behaviors caused by formalin. Opioid receptors in the VP and spinal cord may contribute to these functions.

Selective Activation of NAc D1R-VP/LH Circuits Promotes Reanimation From Sevoflurane Anesthesia in Mice.

BACKGROUND: Emerging evidence has uncovered a vital role of nucleus accumbens (NAc) neurons that express the dopamine D1 receptor (D1R) and its upstream neural circuit in general anesthesia (GA) regulation. However, the underlying downstream neural basis of the modulation of GA emergence by NAc D1R neurons remains unknown. In the present study, we explored the downstream neural mechanism of NAc D1R neurons in the modulation of emergence from sevoflurane GA. METHODS: We traced the axonal projections of NAc D1R neurons using a cell type-specific anterograde tracing method and immunohistochemical techniques in D1R-Cre mice. Optogenetic stimulations combined with electroencephalogram/electromyogram recordings and behavioral tests were used to determine the effects of optogenetic activation of the axonal terminals of NAc D1R neurons on sevoflurane emergence during sevoflurane-induced continuous, steady-state general anesthesia (CSSGA) or burst-suppression oscillations. RESULTS: Labeled efferent fibers of NAc D1R neurons were highly distributed in the ventral pallidum (VP), lateral hypothalamus (LH), and substantia nigra pars compacta. Optogenetic activation of the NAc D1R -VP circuit during CSSGA with sevoflurane induced cortical activation (mean ± standard deviation [SD]; delta power: prestimulation versus during stimulation, 48.7% ± 5.7% vs 35.1% ± 3.3%, P < .0001; beta power: 7.1% ± 2.7% vs 14.2% ± 3.3%, P = .0264) and behavioral emergence, and restored the righting reflex in 66.7% of ChR2 mice. Optogenetic stimulation of the NAc D1R -LH circuit also produced cortical activation (delta power: prestimulation versus during stimulation, 45.0% ± 6.5% vs 36.1% ± 4.6%, P = .0016) and behavioral emergence, and restored the righting reflex in 100% of the ChR2 mice during CSSGA with sevoflurane. Under a sevoflurane-induced burst-suppression state, NAc D1R -VP/LH circuit activation produced evidence of cortical activation (burst-suppression ratio [BSR]: NAc D1R -VP circuit, prestimulation versus during stimulation, 42.4% ± 4.0% vs 26.3% ± 6.0%, P = .0120; prestimulation versus poststimulation, 42.4% ± 4.0% vs 5.9% ± 5.6%, P = .0002; BSR: NAc D1R -LH circuit, prestimulation versus during stimulation, 33.3% ± 13.4% vs 5.1% ± 4.9%, P = .0177; prestimulation vs poststimulation, 33.3% ± 13.4% vs 3.2% ± 4.0%, P = .0105) and behavioral emergence. CONCLUSIONS: Both NAc D1R -VP and NAc D1R -LH circuits are sufficient to promote reanimation from sevoflurane GA by simultaneously inducing cortical and behavioral emergence.

Cadmium-promoted thyroid hormones disruption mediates ROS, inflammation, Aß and Tau proteins production, gliosis, spongiosis and neurodegeneration in rat basal forebrain.

Cadmium (Cd) produces cognition decline following single and repeated treatment, although the complete mechanisms are still unrevealed. Basal forebrain (BF) cholinergic neurons innervate the cortex and hippocampus, regulating cognition. Cd single and repeated exposure induced BF cholinergic neuronal loss, partly through thyroid hormones (THs) disruption, which may cause the cognition decline observed following Cd exposure. However, the mechanisms through which THs disruption mediate this effect remain unknown. To research the possible mechanisms through which Cd-induced THs deficiency may mediate BF neurodegeneration, Wistar male rats were treated with Cd for 1- (1 mg/kg) or 28-days (0.1 mg/kg) with or without triiodothyronine (T3, 40 µg/kg/day). Cd exposure promoted neurodegeneration, spongiosis, gliosis and several mechanisms related to these alterations (increased H202, malondialdehyde, TNF-α, IL-1ß, IL-6, BACE1, Aß and phosphorylated-Tau levels, and decreased phosphorylated-AKT and phosphorylated-GSK-3ß levels). T3 supplementation partially reversed the effects observed. Our results show that Cd induces several mechanisms that may be responsible for the neurodegeneration, spongiosis and gliosis observed in the rats' BF, which are partially mediated by a reduction in THs levels. These data may help to explain the mechanisms through which Cd induces BF neurodegeneration, possibly leading to the cognitive decline observed, providing new therapeutic tools to prevent and treat these damages.

Atrophy of the Cholinergic Basal Forebrain can Detect Presynaptic Cholinergic Loss in Parkinson's Disease.

OBJECTIVES: Structural imaging of the cholinergic basal forebrain may provide a biomarker for cholinergic system integrity that can be used in motor and non-motor outcome studies in Parkinson's disease. However, no prior studies have validated these structural metrics with cholinergic nerve terminal in vivo imaging in Parkinson's disease. Here, we correlate cholinergic basal forebrain morphometry with the topography of vesicular acetylcholine transporter in a large Parkinson's sample. METHODS: [18 F]-Fluoroethoxybenzovesamicol vesicular acetylcholine transporter positron emission tomography was carried out in 101 non-demented people with Parkinson's (76.24% male, mean age 67.6 ± 7.72 years, disease duration 5.7 ± 4.4 years). Subregional cholinergic basal forebrain volumes were measured using magnetic resonance imaging morphometry. Relationships were assessed via volume-of-interest based correlation analysis. RESULTS: Subregional volumes of the cholinergic basal forebrain predicted cholinergic nerve terminal loss, with most robust correlations occurring between the posterior cholinergic basal forebrain and temporofrontal, insula, cingulum, and hippocampal regions, and with modest correlations in parieto-occipital regions. Hippocampal correlations were not limited to the cholinergic basal forebrain subregion Ch1-2. Correlations were also observed in the striatum, thalamus, and brainstem. INTERPRETATION: Cholinergic basal forebrain morphometry is a robust predictor of regional cerebral vesicular acetylcholine transporter bindings, especially in the anterior brain. The relative lack of correlation between parieto-occipital binding and basal forebrain volumes may reflect the presence of more diffuse synaptopathy in the posterior cortex due to etiologies that extend well beyond the cholinergic system. ANN NEUROL 2023;93:991-998.

The amygdala-ventral pallidum pathway contributes to a hypodopaminergic state in the ventral tegmental area during protracted abstinence from chronic cocaine.

BACKGROUND AND PURPOSE: Incubation of craving, the progressive increase in drug seeking over the first weeks of abstinence, is associated with temporal changes during abstinence in the activity of several structures involved in drug-seeking behaviour. Decreases of dopamine (DA) release and DA neuronal activity (hypodopaminergic state) have been reported in the ventral tegmental area (VTA) during cocaine abstinence, but the mechanisms underlying these neuroadaptations are not well understood. We investigated the potential involvement of a VTA inhibiting circuit (basolateral amygdala [BLA]-ventral pallidum [VP] pathway) in the hypodopaminergic state associated with abstinence from chronic cocaine. EXPERIMENTAL APPROACH: In a model of cocaine self-administration, we performed in vivo electrophysiological recordings of DA VTA neurons and BLA neurons from anaesthetised rats during early and protracted abstinence and evaluated the involvement of the BLA-VP pathway using a pharmacological approach. KEY RESULTS: We found significant decreases in VTA DA population activity and significant increases in BLA activity after protracted but not after short-term abstinence from chronic cocaine. The decrease in VTA DA activity was restored by pharmacological inhibition of the activity of either the BLA or the VP, suggesting that these regions exert a negative influence on DA activity. CONCLUSION AND IMPLICATIONS: Our study sheds new lights on neuroadaptations occurring during incubation of craving leading to relapse. In particular, we describe the involvement of the BLA-VP pathway in cocaine-induced decreases of DA activity in the VTA. This study adds important information about the specific brain network dysfunctions underlying hypodopaminergic activity during abstinence.

Cholinergic neurons in the basal forebrain are involved in behavioral abnormalities associated with Cul3 deficiency: Role of prefrontal cortex projections in cognitive deficits.

Loss-of-function mutations of the gene Cul3 have been identified as a risk factor for autism-spectrum disorder (ASD), but the pathogenic mechanisms are not well understood. Conditional Cul3 ablation in cholinergic neurons of mice (ChatCRECul3F/+) recapitulated ASD-like social and sensory gating phenotypes and caused significant cognitive impairments, with diminished activity of cholinergic neurons in the basal forebrain (BF). Chemogenetic inhibition of BF cholinergic neurons in healthy mice induced similar social and cognitive deficits. Conversely, chemogenetic stimulation of BF cholinergic neurons in ChatCRECul3F/+ mice reversed abnormalities in sensory gating and cognition. Cortical hypofunction was also found after ChAT-specific Cul3 ablation and stimulation of cholinergic projections from the BF to the prefrontal cortex (PFC) mitigated cognitive deficits. Overall, we demonstrate that cholinergic dysfunction due to Cul3 deficiency is involved in ASD-like behavioral abnormalities, and that BF cholinergic neurons are particularly critical for cognitive component through their projections to the PFC.

Distribution of vasopressin 1a and oxytocin receptor protein and mRNA in the basal forebrain and midbrain of the spiny mouse (Acomys cahirinus).

The nonapeptide system modulates numerous social behaviors through oxytocin and vasopressin activation of the oxytocin receptor (OXTR) and vasopressin receptor (AVPR1A) in the brain. OXTRs and AVPR1As are widely distributed throughout the brain and binding densities exhibit substantial variation within and across species. Although OXTR and AVPR1A binding distributions have been mapped for several rodents, this system has yet to be characterized in the spiny mouse (Acomys cahirinus). Here we conducted receptor autoradiography and in situ hybridization to map distributions of OXTR and AVPR1A binding and Oxtr and Avpr1a mRNA expression throughout the basal forebrain and midbrain of male and female spiny mice. We found that nonapeptide receptor mRNA is diffuse throughout the forebrain and midbrain and does not always align with OXTR and AVPR1A binding. Analyses of sex differences in brain regions involved in social behavior and reward revealed that males exhibit higher OXTR binding densities in the lateral septum, bed nucleus of the stria terminalis, and anterior hypothalamus. However, no association with gonadal sex was observed for AVPR1A binding. Hierarchical clustering analysis further revealed that co-expression patterns of OXTR and AVPR1A binding across brain regions involved in social behavior and reward differ between males and females. These findings provide mapping distributions and sex differences in nonapeptide receptors in spiny mice. Spiny mice are an excellent organism for studying grouping behaviors such as cooperation and prosociality, and the nonapeptide receptor mapping here can inform the study of nonapeptide-mediated behavior in a highly social, large group-living rodent.

The antipsychotic drug sulpiride in the ventral pallidum paradoxically impairs learning and induces place preference.

Sulpiride, as a D2-like dopamine (DA) receptor (D2R) antagonist, is an important antipsychotic drug in the treatment of schizophrenia. Recently, we have shown that the activation of D2Rs in the ventral pallidum (VP) modulates the activity of the ventral tegmental area (VTA) DAergic neurons. According to our hypothesis, intra-VP sulpiride can influence the motivational and learning processes, pervasively modifying the behavior of examined animals. In the present study, sulpiride was microinjected into the VP of male Wistar rats in three different doses. Morris water maze (MWM) test was applied to investigate the effects of sulpiride on spatial learning, while conditioned place preference (CPP) test was used to examine the potential rewarding effect of the drug. In order to show, whether the animals can associate the rewarding effect with an area which can be recognized only on its spatial location, we introduced a modified version of the CPP paradigm, the spatial CPP test. Our results show that the intra-VP sulpiride dose-dependently impairs learning processes. However, the largest dose of sulpiride induces place preference. Results of the spatial CPP paradigm demonstrate that the animals cannot associate the rewarding effect of the drug with the conditioning area based on its spatial location. In the CPP paradigm, locomotor activity decrease could be observed in the sulpiride-treated rats, likely because of a faster habituation with the conditioning environment. In summary, we can conclude that intra-VP sulpiride has a dual effect: it diminishes the hippocampus-dependent spatial learning processes, in addition, it has a dose-dependent rewarding effect.

Corticotropin Releasing Factor (CRF) Coexpression in GABAergic, Glutamatergic, and GABA/Glutamatergic Subpopulations in the Central Extended Amygdala and Ventral Pallidum of Young Male Primates.

The central extended amygdala (CEA) and ventral pallidum (VP) are involved in diverse motivated behaviors based on rodent models. These structures are conserved, but expanded, in higher primates, including human. Corticotropin releasing factor (CRF), a canonical "stress molecule" associated with the CEA and VP circuitry across species, is dynamically regulated by stress and drugs of abuse and misuse. CRF's effects on circuits critically depend on its colocation with primary "fast" transmitters, making this crucial for understanding circuit effects. We surveyed the distribution and colocalization of CRF-, VGluT2- (vesicular glutamate transporter 2), and VGAT- (vesicular GABA transporter) mRNA in specific subregions of the CEA and VP in young male monkeys. Although CRF-containing neurons were clustered in the lateral central bed nucleus (BSTLcn), the majority were broadly dispersed throughout other CEA subregions, and the VP. CRF/VGAT-only neurons were highest in the BSTLcn, lateral central amygdala nucleus (CeLcn), and medial central amygdala nucleus (CeM) (74%, 73%, and 85%, respectively). In contrast, lower percentages of CRF/VGAT only neurons populated the sublenticular extended amygdala (SLEAc), ventrolateral bed nucleus (BSTLP), and VP (53%, 54%, 17%, respectively), which had higher complements of CRF/VGAT/VGluT2-labeled neurons (33%, 29%, 67%, respectively). Thus, the majority of CRF-neurons at the "poles" (BSTLcn and CeLcn/CeM) of the CEA are inhibitory, while the "extended" BSTLP and SLEAc subregions, and neighboring VP, have a more complex profile with admixtures of "multiplexed" excitatory CRF neurons. CRF's colocalization with its various fast transmitters is likely circuit-specific, and relevant for understanding CRF actions on specific target sites.SIGNIFICANCE STATEMENT The central extended amygdala (CEA) and ventral pallidum (VP) regulate multiple motivated behaviors through differential downstream projections. The stress neuropeptide corticotropin releasing factor (CRF) is enriched in the CEA, and is thought to "set the gain" through modulatory effects on coexpressed primary transmitters. Using protein and transcript assays in monkey, we found that CRF neurons are broadly and diffusely distributed in CEA and VP. CRF mRNA+ neurons colocalize with VGAT (GABA) and VGluT2 (glutamate) mRNAs in different proportions depending on subregion. CRF mRNA was also coexpressed in a subpopulation of VGAT/VGluT2 mRNA ("multiplexed") cells, which were most prominent in the VP and "pallidal"-like parts of the CEA. Heterogeneous CRF and fast transmitter coexpression across CEA/VP subregions implies circuit-specific effects.

Preclinical and randomized clinical evaluation of the p38α kinase inhibitor neflamapimod for basal forebrain cholinergic degeneration.

The endosome-associated GTPase Rab5 is a central player in the molecular mechanisms leading to degeneration of basal forebrain cholinergic neurons (BFCN), a long-standing target for drug development. As p38α is a Rab5 activator, we hypothesized that inhibition of this kinase holds potential as an approach to treat diseases associated with BFCN loss. Herein, we report that neflamapimod (oral small molecule p38α inhibitor) reduces Rab5 activity, reverses endosomal pathology, and restores the numbers and morphology of BFCNs in a mouse model that develops BFCN degeneration. We also report on the results of an exploratory (hypothesis-generating) phase 2a randomized double-blind 16-week placebo-controlled clinical trial (Clinical trial registration: NCT04001517/EudraCT #2019-001566-15) of neflamapimod in mild-to-moderate dementia with Lewy bodies (DLB), a disease in which BFCN degeneration is an important driver of disease expression. A total of 91 participants, all receiving background cholinesterase inhibitor therapy, were randomized 1:1 between neflamapimod 40 mg or matching placebo capsules (taken orally twice-daily if weight <80 kg or thrice-daily if weight >80 kg). Neflamapimod does not show an effect in the clinical study on the primary endpoint, a cognitive-test battery. On two secondary endpoints, a measure of functional mobility and a dementia rating-scale, improvements were seen that are consistent with an effect on BFCN function. Neflamapimod treatment is well-tolerated with no study drug associated treatment discontinuations. The combined preclinical and clinical observations inform on the validity of the Rab5-based pathogenic model of cholinergic degeneration and provide a foundation for confirmatory (hypothesis-testing) clinical evaluation of neflamapimod in DLB.

Resistance of Basal Forebrain Cholinergic Neurons to TDP-43 Proteinopathy in Primary Progressive Aphasia.

Basal forebrain cholinergic neurons (BFCN) display accumulation of neurofibrillary tangles and degeneration in Alzheimer disease and are targets of therapeutic intervention. This study determined vulnerability of BFCN to accumulation of TDP-43 in primary progressive aphasia with TDP-43 proteinopathy (PPA-TDP). Brains from 16 PPA participants with pathologically confirmed TDP-43 proteinopathy, with available paraffin-embedded sections (Group 1), or systematically sampled frozen sections (Group 2), were studied. Immunohistochemistry was performed with an antibody against phosphorylated TDP-43. BFCN were identified by their magnocellular appearance in Nissl preparations. Presence of TDP-43 inclusions and preinclusions in BFCN was determined and quantitative analysis was performed in Group 2. In Group 1, BFCN were completely free of inclusions except for occasional dystrophic neurites. Sparse TDP-43 preinclusions with smooth or granular staining in BFCN were detected. In Group 2, extremely rare TDP-43 intranuclear inclusions were detected in 0.1% of BFCN per section, along with occasional dystrophic neurites. Although sparse, significantly more preinclusions (1.4% of BFCN) were present when compared with inclusions. No hemispheric differences were noted. Small neurons near BFCN contained more preinclusions compared with BFCN. Thus, BFCN in PPA-TDP are resistant to TDP-43 proteinopathy and degeneration, suggesting that cholinergic therapy is unlikely to be effective in this disorder.