Craige C. Wrenn

Galanin transgenic mice display cognitive and neurochemical deficits characteristic of Alzheimer's disease

Galanin is a neuropeptide with multiple inhibitory actions on neurotransmission and memory. In Alzheimers disease (AD), increased galanin-containing fibers hyperinnervate cholinergic neurons within the basal forebrain in association with a decline in cognition. We generated transgenic mice (GAL-tg) that overexpress galanin under the control of the dopamine β-hydroxylase promoter to study the neurochemical and behavioral sequelae of a mouse model of galanin overexpression in AD. Overexpression of galanin was associated with a reduction in the number of identifiable neurons producing acetylcholine in the horizontal limb of the diagonal band. Behavioral phenotyping indicated that GAL-tgs displayed normal general health and sensory and motor abilities; however, GAL-tg mice showed selective performance deficits on the Morris spatial navigational task and the social transmission of food preference olfactory memory test. These results suggest that elevated expression of galanin contributes to the neurochemical and cognitive impairments characteristic of AD.

Galanin GAL-R1 receptor null mutant mice display increased anxiety-like behavior specific to the elevated plus-maze.

The neuropeptide galanin coexists with norepinephrine and serotonin in neural systems mediating emotion. Previous findings suggested that galanin modulates anxiety-related behaviors in rodents. Three galanin receptor subtypes have been cloned; however, understanding their functions has been limited by the lack of galanin receptor subtype-selective ligands. To study the role of the galanin GAL-R1 receptor subtype in mediating anxiety-related behavior, we generated mice with a null mutation in the Galr1 gene. GAL-R1 −/− are viable and show no abnormalities in health, neurological reflexes, motoric functions, or sensory abilities. On a battery of tests for anxiety-like behavior, GAL-R1 −/− showed increased anxiety-like behavior on the elevated plus-maze test. Anxiety-related behaviors on the light/dark exploration, emergence, and open field tests were normal in GAL-R1 −/−. This test-specific anxiety-like phenotype was confirmed in a second, independent cohort of GAL-R1 null mutant mice and +/+ controls. Principal components factor analysis of behavioral scores from 279 mice suggested that anxiety-like behavior on the elevated plus-maze was qualitatively distinct from behavior on other tests in the battery. In addition, exposure to the elevated plus-maze produced a significantly greater neuroendocrine response than exposure to the light/dark exploration test, as analyzed in normal C57BL/6J mice. These behavioral findings in the first galanin receptor null mutant mouse are consistent with the hypothesis that galanin exerts anxiolytic actions via the GAL-R1 receptor under conditions of relatively high stress.

Pharmacological evidence supporting a role for galanin in cognition and affect

1. Galanin is localized in brain pathways involved in both cognition and affect. 2. Galanin has inhibitory actions on a variety of memory tasks including the Morris water maze, delayed nonmatching to position, T-maze delayed alternation, starburst maze, passive avoidance, active avoidance, and spontaneous alternation. 3. Galanin may inhibit learning and memory by inhibiting neurotransmitter release and neuronal firing rate. 4. Two signal transduction mechanisms through which galanin exerts its inhibitory actions are the inhibition of phosphatidyl inositol hydrolysis and the inhibition of adenylate cyclase. 5. Galanin released during periods of burst firing from noradrenergic locus coeruleus terminals in the ventral tegmental area (VTA) may lead to symptoms of depression through inhibition of dopaminergic VTA neurons. 6. Intraventricular galanin has anxiolytic effects in a punished drinking test. Intra-amygdala galanin has anxiogenic effects in a punished drinking test.

Social Transmission of Food Preference in Mice: Methodology and Application to Galanin-Overexpressing Transgenic Mice

Social transmission of food preference (STFP) is a test of olfactory memory that can be used in mice. Confounds in STFP that can lead to misinterpretation of an STFP deficit as a memory impairment include changes in social interaction and olfaction. The issue of changes in social interaction was addressed by evaluating an observer-centric and a demonstrator-centric method for scoring the interaction phase of STFP in mice. The demonstrator-centric method was applied to a line of STFP-impaired, galanin-overexpressing transgenic (GAL-tg). GAL-tg mice were impaired in STFP without deficits in social interaction. In tests of olfactory ability, GAL-tg mice were unimpaired on buried-food and habituation-dishabituation tasks. The current studies describe an expanded method for using STFP in mice and confirm a deficit in olfactory memory in GAL-tg mice.

Learning and memory performance in mice lacking the GAL‐R1 subtype of galanin receptor

The neuropeptide galanin induces performance deficits in a wide range of cognitive tasks in rodents. Three G‐protein‐coupled galanin receptor subtypes, designated GAL‐R1, GAL‐R2 and GAL‐R3, have been cloned. The present study examined the role of GAL‐R1 in cognition by testing mice with a null mutation in Galr1 on several different types of learning and memory tasks. Assessments of general health, neurological reflexes, sensory abilities and motor functions were conducted as control measures. Mutant mice were unimpaired in social transmission of food preference and the Morris water maze. In tests of fear conditioning, mutant mice were unimpaired in a delay version of cued fear conditioning. However, mice homozygous for the null mutation were impaired in a trace version of cued fear conditioning. Mutant mice were unimpaired in contextual fear conditioning, whether training was by the delay or trace protocol. General health, neurological reflexes, sensory abilities and motor functions did not differ across genotypes, indicating that the trace fear conditioning deficit was not an artifact of procedural disabilities. The findings of normal performance on several cognitive tasks and a selective deficit in trace cued fear conditioning in homozygous GAL‐R1 mutant mice are discussed in terms of hypothesized roles of the GAL‐R1 subtype. The generally normal phenotype of GAL‐R1 null mutants supports the use of this line for identification of the receptor subtypes that mediate the cognitive deficits produced by exogenous galanin.

Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate

Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane‐bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N‐acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron–exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT‐PCR analysis of RNA from the brains of –/– mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane‐bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4′‐phosphinicobis‐(butane‐1,3 dicarboxilic acid) (FN6) and 2‐(phosphonomethyl)pentanedioic acid (2‐PMPA) inhibited the residual activity. The IC50 value for 2‐PMPA was about 1 nm for wild‐type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants.

Galanin impairs performance on learning and memory tasks: Findings from galanin transgenic and GAL-R1 knockout mice

Galanin (GAL) impairs performance on cognitive tasks when administered centrally to rats. GAL transgenic (GAL-tg) mice overexpressing endogenous GAL show deficits on the probe trial of the Morris water maze spatial learning task, on the social transmission of food preference olfactory memory task, and on the trace cued fear conditioning emotional learning and memory task. Knockout mice deficient in the GAL-R1 receptor subtype were normal on most memory tasks, while showing a small deficit in trace cued fear conditioning, suggesting a selective role for the GAL-R1 in aversive memories, and implicating other GAL receptor subtypes in spatial learning and olfactory social memory. The growing body of rodent literature implicating excess GAL in cognitive impairment is relevant to the overexpression of GAL in the basal forebrain during the progression of Alzheimers disease.

Performance of galanin transgenic mice in the 5-choice serial reaction time attentional task

The neuropeptide galanin impairs learning and memory in rodents. The mechanism underlying the cognitive effects of galanin may be related to inhibitory effects of galanin on cholinergic transmission. As cholinergic function is thought to modulate sustained attention, the present study examined whether galanin-overexpressing transgenic mice have impairments in sustained attention. Galanin transgenic (GAL-tg) mice and wild-type (WT) littermate controls were trained in a 5-choice serial reaction time task, modified to assess sustained attention. GAL-tg and WT mice performed similarly during acquisition with respect to accuracy, total omissions, and response speed. Attentional mechanisms were challenged by parametric changes including increased event rate, event asynchrony, or decreased stimulus duration. Singly, these challenges did not differentially affect performance between genotypes. Concurrent administration of these challenges, which represents an optimal test of sustained attention, also had similar effects on GAL-tg and WT mice. When stimulus discriminability was reduced by constant illumination of the house light, GAL-tg mice omitted more trials than WT mice, but other measures of performance did not differ by genotype. Moreover, intraventricular injection of galanin in WT mice did not affect sustained attention. These data indicate that previously reported learning and memory effects of galanin are not secondary to attentional dysfunction.