Human Purkinje cells outperform mouse Purkinje cells in dendritic complexity and computational capacity.

Purkinje cells in the cerebellum are among the largest neurons in the brain and have been extensively investigated in rodents. However, their morphological and physiological properties remain poorly understood in humans. In this study, we utilized high-resolution morphological reconstructions and unique electrophysiological recordings of human Purkinje cells ex vivo to generate computational models and estimate computational capacity. An inter-species comparison showed that human Purkinje cell had similar fractal structures but were larger than those of mouse Purkinje cells. Consequently, given a similar spine density (2/µm), human Purkinje cell hosted approximately 7.5 times more dendritic spines than those of mice. Moreover, human Purkinje cells had a higher dendritic complexity than mouse Purkinje cells and usually emitted 2-3 main dendritic trunks instead of one. Intrinsic electro-responsiveness was similar between the two species, but model simulations revealed that the dendrites could process ~6.5 times (n = 51 vs. n = 8) more input patterns in human Purkinje cells than in mouse Purkinje cells. Thus, while human Purkinje cells maintained spike discharge properties similar to those of rodents during evolution, they developed more complex dendrites, enhancing computational capacity.

Compulsive alcohol seeking and relapse: Central role of conditioning factors associated with alleviation of withdrawal states by alcohol.

BACKGROUND AND PURPOSE: Learned associations between environmental stimuli and drugs of abuse represent a major factor in the chronically relapsing nature of drug addiction. In drug dependent subjects these associations must be presumed to include associations linked to reversal of adverse withdrawal states by drug use-"withdrawal-associated learning" (WDL). However, their significance in drug seeking has received little experimental scrutiny. EXPERIMENTAL APPROACH: Using alcohol as a drug of abuse, the behavioural consequences of WDL were investigated in animal models of relapse and compulsive drug seeking by comparing the effects of WD L-associated stimuli versus stimuli associated with alcohol without WDL experience in nondependent and post-dependent rats. Brain sites activated by exposure to the respective stimuli were identified by c-fos immunohistochemistry. KEY RESULTS: (1) WDL-associated stimuli elicited significant alcohol seeking. In rats with WDL experience, stimuli associated with alcohol in the nondependent state no longer elicited robust alcohol seeking. (2) Responding elicited by WDL-associated stimuli, but not stimuli conditioned to alcohol in the nondependent state, was resistant to footshock punishment and increased response effort requirements for presentation of WDL-related stimuli. (3) Stimuli conditioned to alcohol in rats with a dependence but not WDL history did not sustain punished responding or tolerance of increased effort. (4) The central nucleus of the amygdala was identified as a site selectively responsive to WDL stimulus exposure. CONCLUSION AND IMPLICATIONS: Environmental stimuli associated with reversal of adverse withdrawal states by alcohol elicit compulsive-like alcohol seeking and establish WDL as a major, not well-recognized factor, in relapse vulnerability.

Cannabidiol Produces Distinct U-Shaped Dose-Response Effects on Cocaine-Induced Conditioned Place Preference and Associated Recruitment of Prelimbic Neurons in Male Rats.

BACKGROUND: Cannabidiol (CBD) has received attention for the treatment of substance use disorders. In preclinical models of relapse, CBD attenuates drug seeking across several drugs of abuse, including cocaine. However, in these models CBD has not been consistently effective. This inconsistency in CBD effects may be related to presently insufficient information on the full spectrum of CBD dose effects on drug-related behaviors. METHODS: We address this issue by establishing a full dose-response profile of CBD's actions using expression of cocaine-induced conditioned place preference as a model for drug-motivated behavior in male rats and by concurrently identifying dose-dependent effects of CBD on underlying neuronal activation and distinct neuronal phenotypes showing dose-dependent activation changes. Additionally, we established CBD levels in plasma and brain samples. RESULTS: CBD produced linear increases in CBD brain/plasma concentrations but suppressed conditioned place preference in a distinct U-shaped manner. In parallel with its behavioral effects, CBD produced U-shaped suppressant effects on neuronal activation in the prelimbic but not infralimbic cortex or nucleus accumbens core and shell. RNAscope in situ hybridization identified suppression of glutamatergic and GABAergic (gamma-aminobutyric acidergic) signaling in the prelimbic cortex as a possible cellular mechanism for the attenuation of cocaine-induced conditioned place preference by CBD. CONCLUSIONS: The findings extend previous evidence on the potential of CBD in preventing drug-motivated behavior. However, CBD's dose-response profile may have important dosing implications for future clinical applications and may contribute to the understanding of discrepant CBD effects on drug seeking reported in the literature.

Linking drug and food addiction via compulsive appetite.

BACKGROUND AND PURPOSE: 'Food addiction' is the subject of intense public and research interest. However, this nosology based on neurobehavioural similarities among obese individuals, patients with eating disorders and those with substance use disorders (drug addiction) remains controversial. We thus sought to determine which aspects of disordered eating are causally linked to preclinical models of drug addiction. We hypothesized that extensive drug histories, known to cause addiction-like brain changes and drug motivation in rats, would also cause addiction-like food motivation. EXPERIMENTAL APPROACH: Rats underwent extensive cocaine, alcohol, caffeine or obesogenic diet histories and were subsequently tested for punishment-resistant food self-administration or 'compulsive appetite', as a measure of addiction-like food motivation. KEY RESULTS: Extensive cocaine and alcohol (but not caffeine) histories caused compulsive appetite that persisted long after the last drug exposure. Extensive obesogenic diet histories also caused compulsive appetite, although neither cocaine nor alcohol histories caused excess calorie intake and bodyweight during abstinence. Hence, compulsive appetite and obesity appear to be dissociable, with the former sharing common mechanisms with preclinical drug addiction models. CONCLUSION AND IMPLICATIONS: Compulsive appetite, as seen in subsets of obese individuals and patients with binge-eating disorder and bulimia nervosa (eating disorders that do not necessarily result in obesity), appears to epitomize 'food addiction'. Because different drug and obesogenic diet histories caused compulsive appetite, overlapping dysregulations in the reward circuits, which control drug and food motivation independently of energy homeostasis, may offer common therapeutic targets for treating addictive behaviours across drug addiction, eating disorders and obesity.

Reorganization of longitudinal compartments in the laterally protruding paraflocculus of the postnatal mouse cerebellum.

The paraflocculus and the neighboring smaller flocculus form a remarkable protrusion in the ventrolateral aspect of the mouse cerebellum, in which the longitudinal compartments are conspicuously oriented perpendicularly to the sagittal plane. The developmental process of such anatomical arrangements in these lobules has not been fully clarified. Here, we used the genetic tractability of pcdh10-lacZ knock-in (OL-KO), IP 3 R1-nls-lacZ transgenic (1NM13) and Gpr26cre-Ai9-AldocV mice to track the development of compartments and examined local longitudinal orientation of Purkinje cells within the paraflocculus and flocculus. We observed a distinct pcdh10-positive (pcdh10+) compartment in the flocculus, whereas the paraflocculus and other lobules had a continuous paravermal pcdh10+ compartment, in the embryonic OL-KO cerebellum. During the first postnatal week, the parafloccular pcdh10+ compartment shifted laterally to the most lateral edge in the caudal part of the protruding paraflocculus. Although the most medial edge of the parafloccular pcdh10+ compartment remained in the nonprotruding part of the paraflocculus, it was disrupted from the originally continuous pcdh10+ compartment in the copula pyramidis. The local longitudinal orientation changed gradually along with the mediolateral extent of the copula pyramidis, almost becoming perpendicular to the sagittal plane in the laterally connected paraflocculus in the adult cerebellum. This rotational change in orientation was derived from the short U-shaped embryonic cerebellum, in which the surfaces of the flocculus and paraflocculus were oriented laterally. These results indicated that the peculiar compartmental organization of the paraflocculus originates from the embryonic common hemispheric compartmental organization and shaped by the significant reorganization process in the first postnatal week.

Anti-relapse neurons in the infralimbic cortex of rats drive relapse-suppression by drug omission cues.

Drug addiction is a chronic relapsing disorder of compulsive drug use. Studies of the neurobehavioral factors that promote drug relapse have yet to produce an effective treatment. Here we take a different approach and examine the factors that suppress-rather than promote-relapse. Adapting Pavlovian procedures to suppress operant drug response, we determined the anti-relapse action of environmental cues that signal drug omission (unavailability) in rats. Under laboratory conditions linked to compulsive drug use and heightened relapse risk, drug omission cues suppressed three major modes of relapse-promotion (drug-predictive cues, stress, and drug exposure) for cocaine and alcohol. This relapse-suppression is, in part, driven by omission cue-reactive neurons, which constitute small subsets of glutamatergic and GABAergic cells, in the infralimbic cortex. Future studies of such neural activity-based cellular units (neuronal ensembles/memory engram cells) for relapse-suppression can be used to identify alternate targets for addiction medicine through functional characterization of anti-relapse mechanisms.

Regional differences in Purkinje cell morphology in the cerebellar vermis of male mice.

Regional differences in dendritic architecture can influence connectivity and dendritic signal integration, with possible consequences for neuronal computation. In the cerebellum, analyses of Purkinje cells (PCs), which are functionally critical as they provide the sole output of the cerebellar cortex, have suggested that the cerebellar cortex is not uniform in structure as traditionally assumed. However, the limitations of traditional staining methods and microscopy capabilities have presented difficulties in investigating possible local variations in PC morphology. To address this question, we used male mice expressing green fluorescent protein selectively in PCs. Using Neurolucida 360 with confocal image stacks, we reconstructed dendritic arbors of PCs residing in lobule V (anterior) and lobule IX (posterior) of the vermis. We then analyzed morphologies of individual arbors and the structure of the assembled "jungle," comparing these features across anatomical locations and age groups. Strikingly, we found that in lobule IX, half of the reconstructed PCs had two primary dendrites emanating from their soma, whereas fewer than a quarter showed this characteristic in lobule V. Furthermore, PCs in lobule V showed more efficient spatial occupancy compared to lobule IX, as well as greater packing density and increased arbor overlap in the adult. When analyzing complete ensembles of PC arbors, we also observed "hot spots" of increased dendritic density in lobule V, whereas lobule IX showed a more homogeneous spread of dendrites. These differences suggest that input patterns and/or physiology of PCs could likewise differ along the vermis, with possible implications for cerebellar function.

Lobular homology in cerebellar hemispheres of humans, non-human primates and rodents: a structural, axonal tracing and molecular expression analysis.

Comparative neuroanatomy provides insights into the evolutionary functional adaptation of specific mammalian cerebellar lobules, in which the lobulation pattern and functional localization are conserved. However, accurate identification of homologous lobules among mammalian species is challenging. In this review, we discuss the inter-species homology of crus I and II lobules which occupy a large volume in the posterior cerebellar hemisphere, particularly in humans. Both crus I/II in humans are homologous to crus I/II in non-human primates, according to Paxinos and colleagues; however, this area has been defined as crus I alone in non-human primates, according to Larsell and Brodal. Our neuroanatomical analyses in humans, macaques, marmosets, rats, and mice demonstrate that both crus I/II in humans are homologous to crus I/II or crus I alone in non-human primates, depending on previous definitions, and to crus I alone in rodents. Here, we refer to the region homologous to human crus I/II lobules as "ansiform area (AA)" across animals. Our results show that the AA's olivocerebellar climbing fiber and Purkinje cell projections as well as aldolase C gene expression patterns are both distinct and conserved in marmosets and rodents. The relative size of the AA, as represented by the AA volume fraction in the whole cerebellum was 0.34 in human, 0.19 in macaque, and approximately 0.1 in marmoset and rodents. These results indicate that the AA reflects an evolutionarily conserved structure in the mammalian cerebellum, which is characterized by distinct connectivity from neighboring lobules and a massive expansion in skillful primates.

Cerebellar sub-divisions differ in exercise-induced plasticity of noradrenergic axons and in their association with resilience to activity-based anorexia.

The vermis or "spinocerebellum" receives input from the spinal cord and motor cortex for controlling balance and locomotion, while the longitudinal hemisphere region or "cerebro-cerebellum" is interconnected with non-motor cortical regions, including the prefrontal cortex that underlies decision-making. Noradrenaline release in the cerebellum is known to be important for motor plasticity but less is known about plasticity of the cerebellar noradrenergic (NA) system, itself. We characterized plasticity of dopamine ß-hydroxylase-immunoreactive NA fibers in the cerebellum of adolescent female rats that are evoked by voluntary wheel running, food restriction (FR) or by both, in combination. When 8 days of wheel access was combined with FR during the last 4 days, some responded with excessive exercise, choosing to run even during the hours of food access: this exacerbated weight loss beyond that due to FR alone. In the vermis, exercise, with or without FR, shortened the inter-varicosity intervals and increased varicosity density along NA fibers, while excessive exercise, due to FR, also shortened NA fibers. In contrast, the hemisphere required the FR-evoked excessive exercise to evoke shortened inter-varicosity intervals along NA fibers and this change was exhibited more strongly by rats that suppressed the FR-evoked excessive exercise, a behavior that minimized weight loss. Presuming that shortened inter-varicosity intervals translate to enhanced NA release and synthesis of norepinephrine, this enhancement in the cerebellar hemisphere may contribute towards protection of individuals from the life-threatening activity-based anorexia via relays with higher-order cortical areas that mediate the animal's decision to suppress the innate FR-evoked hyperactivity.

Dendritic diameters affect the spatial variability of intracellular calcium dynamics in computer models.

There is growing interest in understanding calcium dynamics in dendrites, both experimentally and computationally. Many processes influence these dynamics, but in dendrites there is a strong contribution of morphology because the peak calcium levels are strongly determined by the surface to volume ratio (SVR) of each branch, which is inversely related to branch diameter. In this study we explore the predicted variance of dendritic calcium concentrations due to local changes in dendrite diameter and how this is affected by the modeling approach used. We investigate this in a model of dendritic calcium spiking in different reconstructions of cerebellar Purkinje cells and in morphological analysis of neocortical and hippocampal pyramidal neurons. We report that many published models neglect diameter-dependent effects on calcium concentration and show how to implement this correctly in the NEURON simulator, both for phenomenological pool based models and for implementations using radial 1D diffusion. More detailed modeling requires simulation of 3D diffusion and we demonstrate that this does not dissipate the local concentration variance due to changes of dendritic diameter. In many cases 1D diffusion of models of calcium buffering give a good approximation provided an increased morphological resolution is implemented.

Comparative morphology of dendritic arbors in populations of Purkinje cells in mouse sulcus and apex.

Foliation divides the mammalian cerebellum into structurally distinct subdivisions, including the concave sulcus and the convex apex. Purkinje cell (PC) dendritic morphology varies between subdivisions and changes significantly ontogenetically. Since dendritic morphology both enables and limits sensory-motor circuit function, it is important to understand how neuronal architectures differ between brain regions. This study employed quantitative confocal microcopy to reconstruct dendritic arbors of cerebellar PCs expressing green fluorescent protein and compared arbor morphology between PCs of sulcus and apex in young and old mice. Arbors were digitized from high z-resolution (0.25 µm) image stacks using an adaptation of Neurolucida's (MBF Bioscience) continuous contour tracing tool, designed for drawing neuronal somata. Reconstructed morphologies reveal that dendritic arbors of sulcus and apex exhibit profound differences. In sulcus, 72% of the young PC population possesses two primary dendrites, whereas in apex, only 28% do. Spatial constraints in the young sulcus cause significantly more dendritic arbor overlap than in young apex, a distinction that disappears in adulthood. However, adult sulcus PC arbors develop a greater number of branch crossings. These results suggest developmental neuronal plasticity that enables cerebellar PCs to attain correct functional adult architecture under different spatial constraints.

Stochastic calcium mechanisms cause dendritic calcium spike variability.

Bursts of dendritic calcium spikes play an important role in excitability and synaptic plasticity in many types of neurons. In single Purkinje cells, spontaneous and synaptically evoked dendritic calcium bursts come in a variety of shapes with a variable number of spikes. The mechanisms causing this variability have never been investigated thoroughly. In this study, a detailed computational model using novel simulation routines is applied to identify the roles that stochastic ion channels, spatial arrangements of ion channels, and stochastic intracellular calcium have toward producing calcium burst variability. Consistent with experimental recordings from rats, strong variability in the burst shape is observed in simulations. This variability persists in large model sizes in contrast to models containing only voltage-gated channels, where variability reduces quickly with increase of system size. Phase plane analysis of Hodgkin-Huxley spikes and of calcium bursts identifies fluctuation in phase space around probabilistic phase boundaries as the mechanism determining the dependence of variability on model size. Stochastic calcium dynamics are the main cause of calcium burst fluctuations, specifically the calcium activation of mslo/BK-type and SK2 channels. Local variability of calcium concentration has a significant effect at larger model sizes. Simulations of both spontaneous and synaptically evoked calcium bursts in a reconstructed dendrite show, in addition, strong spatial and temporal variability of voltage and calcium, depending on morphological properties of the dendrite. Our findings suggest that stochastic intracellular calcium mechanisms play a crucial role in dendritic calcium spike generation and are therefore an essential consideration in studies of neuronal excitability and plasticity.

Endogenous GluR1-containing AMPA receptors translocate to asymmetric synapses in the lateral amygdala during the early phase of fear memory formation: an electron microscopic immunocytochemical study.

Although glutamate receptor 1 (GluR1)-containing α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (GluR1-AMPARs) are implicated in synaptic plasticity, it has yet to be demonstrated whether endogenous GluR1-AMPARs undergo activity-dependent trafficking in vivo to synapses to support short-term memory (STM) formation. The paradigm of pavlovian fear conditioning (FC) can be used to address this question, because a discrete region-the lateral amygdala (LA)-has been shown unambiguously to be necessary for the formation of the associative memory between a neutral stimulus (tone [CS]) and a noxious stimulus (foot shock [US]). Acquisition of STM for FC can occur even in the presence of protein synthesis inhibitors, indicating that redistribution of pre-existing molecules to synaptic junctions underlies STM. We employed electron microscopic immunocytochemistry to evaluate alterations in the distribution of endogenous AMPAR subunits at LA synapses during the STM phase of FC. Rats were sacrificed 40 minutes following three CS-US pairings. In the LA of paired animals, relative to naïve animals, the proportion of GluR1-AMPAR-labeled synapses increased 99% at spines and 167% in shafts. In the LA of unpaired rats, for which the CS was never associated with the US, GluR1 immunoreactivity decreased 84% at excitatory shaft synapses. GluR2/3 immunoreactivity at excitatory synapses did not change detectably following paired or unpaired conditioning. Thus, the early phase of FC involves rapid redistribution specifically of the GluR1-AMPARs to the postsynaptic membranes in the LA, together with the rapid translocation of GluR1-AMPARs from remote sites into the spine head cytoplasm, yielding behavior changes that are specific to stimulus contingencies.

Increased levels of NMDA receptor NR2A subunits at pre- and postsynaptic sites of the hippocampal CA1: an early response to conditional double knockout of presenilin 1 and 2.

Greater than 90% of familial Alzheimer's disease (AD) is linked to mutations of presenilin (PS), and the loss of PS function altogether within mouse brains by conditional double knockout of the PS 1 and 2 genes (PS-cDKO) leads to age-dependent emergence of AD phenotypes, including neurodegeneration and reduced synaptic plasticity in the hippocampal CA1. The goal of our study was to identify the ultrastructural and molecular changes at synapses in the hippocampal CA1 of this PS-cDKO mouse model of AD. We examined the asymmetric (excitatory) synapses formed on apical dendrites of CA1 pyramidal neurons at 2 months postnatal, an age when AD-like symptoms emerge but brain morphology, as assessed by light microscopy, is still normal. Our quantitative electron microscopic analyses confirm that PS-cDKO hippocampi at 2 months postnatal do not yet exhibit synapse losses or spine size alterations. However, immunocytochemistry reveals that the same region exhibits a 28% increase in the proportion of spines labeled for the NR2A subunits of NMDA receptors (NMDAR), with a 31% increase specifically at postsynaptic densities and a concomitant reduction of these subunits at nonsynaptic sites within spine heads. In contrast, no change in levels or the distribution pattern of NR2B subunit levels were detected within spine heads. Presynaptically, NR2A levels are elevated at axo-spinous junctions and these may contribute to the timing-dependent, long-term depression. These observations point to an early-onset trapping of NMDAR at synapses that are subtle but may underlie the reduced synaptic plasticity at 2 months of age and excitotoxicity at later stages.