John K. Robinson

Anandamide, an endogenous ligand of the cannabinoid receptor, induces hypomotility and hypothermia in vivo in rodents.

Anandamide (arachidonylethanolamide), an arachidonic acid derivative isolated from the porcine brain, displays binding characteristics indicative of an endogenous ligand for the cannabinoid receptor. The functional activity of anandamide was tested in vivo using behavioral and physiological paradigms in laboratory rodents. At IP doses from 2 to 20 mg/kg in mice, anandamide significantly decreased spontaneous motor activity in a Digiscan open field. Rectal body temperature significantly decreased at doses of 10 and 20 mg/kg in rats. At doses from 0.03 to 30 mg/kg, anandamide had no significant effect on chow consumption in ad lib fed rats. Over the dose range of 2-20 mg/kg, anandamide did not show anxiolytic properties in the mouse light<-->dark exploration model of anxiety. Over the dose range of 0.3-3 mg/kg, anandamide had no effect on choice accuracy or session duration in the delayed nonmatching to sample memory task (DNMTS) in rats. These results demonstrate that anandamide has biological and behavioral effects in awake rodents, some of which are similar to the reported actions of THC.

Minocycline reduces microglial activation and improves behavioral deficits in a transgenic model of cerebral microvascular amyloid.

Cerebral microvascular amyloid β protein (Aβ) deposition and associated neuroinflammation is increasingly recognized as an important component leading to cognitive impairment in Alzheimers disease and related cerebral amyloid angiopathy disorders. Transgenic mice expressing the vasculotropic Dutch/Iowa (E693Q/D694N) mutant human Aβ precursor protein in brain (Tg-SwDI) accumulate abundant cerebral microvascular fibrillar amyloid deposits and exhibit robust neuroinflammation. In the present study, we investigated the effect of the anti-inflammatory drug minocycline on Aβ accumulation, neuroinflammation, and behavioral deficits in Tg-SwDI mice. Twelve-month-old mice were treated with saline or minocycline by intraperitoneal injection every other day for a total of 4 weeks. During the final week of treatment, the mice were tested for impaired learning and memory. Brains were then harvested for biochemical and immunohistochemical analysis. Minocycline treatment did not alter the cerebral deposition of Aβ or the restriction of fibrillar amyloid to the cerebral microvasculature. Similarly, minocycline-treated Tg-SwDI mice exhibited no change in the levels of total Aβ, the ratios of Aβ40 and Aβ42, or the amounts of soluble, insoluble, or oligomeric Aβ compared with the saline-treated control Tg-SwDI mice. In contrast, the numbers of activated microglia and levels of interleukin-6 were significantly reduced in minocycline-treated Tg-SwDI mice compared with saline-treated Tg-SwDI mice. In addition, there was a significant improvement in behavioral performance of the minocycline-treated Tg-SwDI mice. These finding suggest that anti-inflammatory treatment targeted for cerebral microvascular amyloid-induced microglial activation can improve cognitive deficits without altering the accumulation and distribution of Aβ.

Galanin Antagonists Block Galanin‐induced Feeding in the Hypothalamus and Amygdala of the Rat

Galanin significantly increased food intake when microinjected into the region of the central nucleus of the amygdala as well as into the paraventricular nucleus of the hypothalamus. In the amygdala this effect was specific to feeding; no change in grooming, resting, or other behaviour was observed after galanin treatment. These results provide evidence that the amygdala may be an important site in the mediation of galanin‐induced feeding. The galanin receptor antagonists, C7 and M40, antagonized galanin‐induced feeding, while having no effect alone on food consumption in free‐feeding rats. These new galanin receptor antagonists provide useful tools for further investigating the role of endogenous galanin in the regulation of feeding.

A complement–microglial axis drives synapse loss during virus-induced memory impairment

Over 50% of patients who survive neuroinvasive infection with West Nile virus (WNV) exhibit chronic cognitive sequelae. Although thousands of cases of WNV-mediated memory dysfunction accrue annually, the mechanisms responsible for these impairments are unknown. The classical complement cascade, a key component of innate immune pathogen defence, mediates synaptic pruning by microglia during early postnatal development. Here we show that viral infection of adult hippocampal neurons induces complement-mediated elimination of presynaptic terminals in a murine WNV neuroinvasive disease model. Inoculation of WNV-NS5-E218A, a WNV with a mutant NS5(E218A) protein leads to survival rates and cognitive dysfunction that mirror human WNV neuroinvasive disease. WNV-NS5-E218A-recovered mice (recovery defined as survival after acute infection) display impaired spatial learning and persistence of phagocytic microglia without loss of hippocampal neurons or volume. Hippocampi from WNV-NS5-E218A-recovered mice with poor spatial learning show increased expression of genes that drive synaptic remodelling by microglia via complement. C1QA was upregulated and localized to microglia, infected neurons and presynaptic terminals during WNV neuroinvasive disease. Murine and human WNV neuroinvasive disease post-mortem samples exhibit loss of hippocampal CA3 presynaptic terminals, and murine studies revealed microglial engulfment of presynaptic terminals during acute infection and after recovery. Mice with fewer microglia (Il34−/− mice with a deficiency in IL-34 production) or deficiency in complement C3 or C3a receptor were protected from WNV-induced synaptic terminal loss. Our study provides a new murine model of WNV-induced spatial memory impairment, and identifies a potential mechanism underlying neurocognitive impairment in patients recovering from WNV neuroinvasive disease.

MeCP2 is critical for maintaining mature neuronal networks and global brain anatomy during late stages of postnatal brain development and in the mature adult brain

Mutations in the X-linked gene, methyl-CpG binding protein 2 (Mecp2), underlie a wide range of neuropsychiatric disorders, most commonly, Rett Syndrome (RTT), a severe autism spectrum disorder that affects approximately one in 10,000 female live births. Because mutations in the Mecp2 gene occur in the germ cells with onset of neurological symptoms occurring in early childhood, the role of MeCP2 has been ascribed to brain maturation at a specific developmental window. Here, we show similar kinetics of onset and progression of RTT-like symptoms in mice, including lethality, if MeCP2 is removed postnatally during the developmental stage that coincides with RTT onset, or adult stage. For the first time, we show that brains that lose MeCP2 at these two different stages are actively shrinking, resulting in higher than normal neuronal cell density. Furthermore, we show that mature dendritic arbors of pyramidal neurons are severely retracted and dendritic spine density is dramatically reduced. In addition, hippocampal astrocytes have significantly less complex ramified processes. These changes accompany a striking reduction in the levels of several synaptic proteins, including CaMKII α/β, AMPA, and NMDA receptors, and the synaptic vesicle proteins Vglut and Synapsin, which represent critical modifiers of synaptic function and dendritic arbor structure. Importantly, the mRNA levels of these synaptic proteins remains unchanged, suggesting that MeCP2 likely regulates these synaptic proteins post-transcriptionally, directly or indirectly. Our data suggest a crucial role for MeCP2 in post-transcriptional regulation of critical synaptic proteins involved in maintaining mature neuronal networks during late stages of postnatal brain development.

Changes in hippocampal morphology following chronic treatment with the synthetic cannabinoid WIN 55,212-2.

Learning and memory are often correlated with cellular changes within the hippocampus, and drugs or environmental factors which affect learning and memory will thus often induce observable morphological changes in this structure. Like tetrahydrocannabinol (THC) itself, many synthetic cannabinoids such as the CB-1 receptor agonist WIN 55,212-2 will induce learning and memory changes. In the current study, we investigate whether or not these changes could be related to structural changes within the hippocampus. Adult male Sprague-Dawley rats were injected twice daily (12:00 and 0:00 h) subcutaneously with WIN 55,212-2 (2.0 mg/kg) in DMSO or DMSO for 21 days. On day 22, animals were perfused and stained immunochemically for the dendritic marker MAP-2, or with cresyl violet. Morphometric analysis showed dendritic rearrangement with increased staining of MAP-2 in CA3 and the lower blade of the dentate gyrus. However, a loss of staining was observed in CA1. Counting of cresyl violet stained sections showed an apparent increase in granule cell number in the lower blade of the dentate gyrus. This work shows the potential for cannabinoids to influence hippocampal morphology. The pattern of changes may be similar to that seen after ischemic or toxic damage, but may be opposite to changes seen in stress.

Gonadectomy in adult life increases tyrosine hydroxylase immunoreactivity in the prefrontal cortex and decreases open field activity in male rats

The prefrontal cortices in rats participate in a range of cognitive, emotional, and locomotor functions that are dependent on its rich catecholamine innervation. Sex differences identified in many of these functions suggest that the prefrontal cortex is also influenced by gonadal hormones. Previous studies have shown that prefrontal catecholamines can be modified by changes in the hormone environment in developing animals. The present analyses, carried out in male rats gonadectomized as adults, with and without supplementation with testosterone proprionate, and examined at intervals from two days to 10 weeks after surgery, revealed that both the anatomical organization of prefrontal catecholamine afferents, and a behavioral measure sensitive to their selective lesioning remain highly responsive to changes in testicular hormones in adulthood. Thus, gonadectomy in adult male rats rapidly led to a large but transient decrease in the density of tyrosine hydroxylase immunoreactivity in all layers of the dorsal anterior cingulate cortex. This was followed by a sustained period in which immunoreactivity in the supragranular layers returned to levels that were just below normal (between 72 and 79% of normal), and labeling in deep laminae stabilized at considerably elevated innervation densities (approximately 150% of normal). Neither the acute decrease nor the chronic over-innervation characteristic of gonadectomized animals was observed in rats that were gonadectomized and supplemented with testosterone proprionate. Open field activity assessed along a corresponding 10 week timeline showed that gonadectomized animals were significantly less active than hormonally intact controls, a behavioral pattern opposite to the hyperactivity which persists following prefrontal dopamine lesions. Gonadectomized animals supplemented with testosterone proprionate, on the other hand, had open field scores that were not significantly different from controls. Taken together, these findings indicate that the adult hormone environment provides a significant, and seemingly functionally significant influence over the catecholamine innervation of the rat prefrontal cortex. Such lifelong responsiveness of the prefrontal cortical catecholamines to circulating hormones suggests that gonadal steroids are an active component of the biology of normal adult cognition, and may also have relevance for cortical dysfunction in disorders such as schizophrenia which are not only strongly tied to the catecholamines, but exhibit considerable biases among men and women as well.

Effects of gonadectomy on performance in operant tasks measuring prefrontal cortical function in adult male rats

Previous studies have shown that gonadectomy in adult male rats influences the acquisition and performance of spatial and other working memory tasks that depend in part on the medial prefrontal cortex and its dopamine innervation. Stimulated by previous findings that gonadectomy alters dopamine axon density in not only medial but several other prefrontal fields, the present studies asked whether gonadectomy might also broadly impact dopamine-dependent prefrontal functions, and whether these effects bore any relation to hormone modulation of mesoprefrontal dopamine afferents. Specifically, control, gonadectomized, and gonadectomized rats given estradiol or testosterone propionate were tested on a series of operant tasks that together measured medial prefrontal functions of spatial working memory, impulsivity and extradimensional set shifting and orbital prefrontal functions of reversal learning/perseveration and motivation. Afterwards, animals were sacrificed, their bulbospongiosus muscles were removed and weighed, their brains were processed for immunocytochemistry for the dopamine-synthesizing enzyme tyrosine hydroxylase, and axon densities were measured in orbital and medial prefrontal fields. Statistical evaluations of group effects on behavior and regression analyses comparing individual performance with muscle weights and axon density measures revealed androgen-reversible effects of gonadectomy on acquisition of spatial working memory and extradimensional set shifting that were correlated with bulbospongiosus weight and medial prefrontal dopamine axon density, estrogen-sensitive influences of gonadectomy on motivation and response withholding that were correlated with bulbospongiosus weight but not with dopamine innervation, and still other prefrontal functions, i.e., impulsivity, reversal learning, that were insensitive to gonadectomy and unrelated to gonadectomy-induced changes in muscle weight or prefrontal dopamine innervation.

Chronic spinal hemisection in rats induces a progressive decline in transmission in uninjured fibers to motoneurons.

Although most spinal cord injuries are anatomically incomplete, only limited functional recovery has been observed in people and rats with partial lesions. To address why surviving fibers cannot mediate more complete recovery, we evaluated the physiological and anatomical status of spared fibers after unilateral hemisection (HX) of thoracic spinal cord in adult rats. We made intracellular and extracellular recordings at L5 (below HX) in response to electrical stimulation of contralateral white matter above (T6) and below (L1) HX. Responses from T6 displayed reduced amplitude, increased latency and elevated stimulus threshold in the fibers across from HX, beginning 1-2 weeks after HX. Ultrastructural analysis revealed demyelination of intact axons contralateral to the HX, with a time course similar to the conduction changes. Behavioral studies indicated partial recovery which arrested when conduction deficits began. In conclusion, this study is the first demonstration of the delayed decline of transmission through surviving axons to individual lumbar motoneurons during chronic stage of incomplete spinal cord injury in adult rats. These findings suggest a chronic pathological state in intact fibers and necessity for prompt treatment to minimize it.

Galanin Inhibits Performance on Rodent Memory Tasks

Abstract: Central administration of galanin produces performance deficits on a variety of rodent learning and memory tasks. Galanin impairs acquisition and/or retention of the Morris water task, delayed nonmatching to position, T‐maze delayed alternation, starburst radial maze, and passive avoidance in normal rats. A primary site of action is the ventral hippocampus, with an additional modulatory site in the medial septum‐diagonal band. The behavioral actions of galanin at rat septohippo‐campal sites mediating cognitive processes are consistent with previous reports of inhibitory actions of galanin on acetylcholine release and cholinergically activated transduction at the M1 muscarinic receptor in rat hippocampus. The peptidergic galanin receptor antagonist M40 blocks the inhibitory actions of galanin on memory tasks. Treatment combinations of M40 with an M1 agonist, TZTP, improves performance on delayed nonmatching to position, in rats with 192IgG‐saporin‐induced cholinergic lesions of basal forebrain neurons. Nonpeptide, bioavailable, subtype‐selective galanin receptor antagonists may provide tools to test the hypothesis that antagonism of endogenous galanin, which is overexpressed in the basal forebrain in Alzheimers patients, can contribute to the alleviation of the cognitive deficits associated with Alzheimers disease.