Patricia Szot

Compensatory Changes in the Noradrenergic Nervous System in the Locus Ceruleus and Hippocampus of Postmortem Subjects with Alzheimer's Disease and Dementia with Lewy Bodies

In Alzheimers disease (AD), there is a significant loss of locus ceruleus (LC) noradrenergic neurons. However, functional and anatomical evidence indicates that the remaining noradrenergic neurons may be compensating for the loss. Because the noradrenergic system plays an important role in learning and memory, it is important to determine whether compensation occurs in noradrenergic neurons in the LC and hippocampus of subjects with AD or a related dementing disorder, dementia with Lewy bodies (DLB). We observed profound neuronal loss in the LC in AD and DLB subjects with three major changes in the noradrenergic system consistent with compensation: (1) an increase in tyrosine hydroxylase (TH) mRNA expression in the remaining neurons; (2) sprouting of dendrites into peri-LC dendritic zone, as determined by α2-adrenoreceptors (ARs) and norepinephrine transporter binding sites; and (3) sprouting of axonal projections to the hippocampus as determined by α2-ARs. In AD and DLB subjects, the postsynaptic α1-ARs were normal to elevated. Expression of α1A- and α2A-AR mRNA in the hippocampus of AD and DLB subjects were not altered, but expression of α1D- and α2C-AR mRNA was significantly reduced in the hippocampus of AD and DLB subjects. Therefore, in AD and DLB subjects, there is compensation occurring in the remaining noradrenergic neurons, but there does appear to be a loss of specific AR in the hippocampus. Because changes in these noradrenergic markers in AD versus DLB subjects were similar (except neuronal loss and the increase in TH mRNA were somewhat greater in DLB subjects), the presence of Lewy bodies in addition to plaques and tangles in DLB subjects does not appear to further affect the noradrenergic compensatory changes.

The role of catecholamines in seizure susceptibility: new results using genetically engineered mice.

The catecholamines norepinephrine and dopamine are abundant in the CNS, and modulate neuronal excitability via G-protein-coupled receptor signaling. This review covers the history of research concerning the role of catecholamines in modulating seizure susceptibility in animal models of epilepsy. Traditionally, most work on this topic has been anatomical, pharmacological, or physiological in nature. However, the recent advances in transgenic and knockout mouse technology provide new tools to study catecholamines and their roles in seizure susceptibility. New results from genetically engineered mice with altered catecholamine signaling, as well as possibilities for future experiments, are discussed.

Intraventricular insulin increases dopamine transporter mRNA in rat VTA/substantia nigra.

The hormone insulin can down-regulate the function and synthesis of the re-uptake transporter for norepinephrine (NET) in vivo and in vitro. In the present study we tested whether this action of insulin is generalized to another member of the catecholamine transporter family. We determined the level of dopamine transporter (DAT) mRNA expression in the ventral tegmental area (VTA)/substantia nigra compacta (SNc) of rats which were chronically treated with vehicle or insulin via the third cerebral ventricle (i.c.v.). DAT mRNA was significantly elevated in the VTA/SNc of rats treated with insulin, as compared with levels in vehicle-treated rats. This is in contrast to our previous observation that i.c.v. insulin decreases NET mRNA in the rat locus coeruleus, and suggests that insulin may have differential and specific modulatory effects on CNS catecholaminergic pathways.

Food Deprivation Decreases mRNA and Activity of the Rat Dopamine Transporter

We have hypothesized that the midbrain dopamine (DA) neurons are a target for insulin action in the central nervous system (CNS). In support of this hypothesis, we have previously demonstrated that direct intracerebroventricular infusion of insulin results in an increase in mRNA levels for the DA reuptake transporter (DAT). In this study, 24- to 36-hour food deprivation was used as a model of decreased CNS insulin levels, to test whether DAT mRNA levels, DAT protein concentration or DAT functional activity would be decreased. DAT mRNA levels, assessed by in situ hybridization, were significantly decreased in the ventral tegmental area/substantia nigra pars compacta (VTA/SNc) (77 ± 7% of controls, p < 0.05) of food-deprived (hypoinsulinemic) rats. Binding of a specific high-affinity DAT ligand (125I-RTI-121) to membranes from brain regions of fasted or free-feeding rats provided an estimate of DAT protein, which was unchanged in both of the major terminal projection fields, the striatum and nucleus accumbens (NAc). In addition, we utilized the rotating disk electrode voltametry technique to assess possible changes in the function of the DAT in fasting (hypoinsulinemic) rats. The Vmax of DA uptake was significantly decreased (87 ± 7% of control, p < 0.05), without a change in the Km of uptake, in striatum from fasted rats. In vitro incubation with a physiological concentration (1 nM) of insulin resulted in an increase of striatal DA uptake to control levels. We conclude that striatal DAT function can be modulated by fasting and nutritional status, with a contribution by insulin.

Insulin reduces norepinephrine transporter mRNA in vivo in rat locus coeruleus.

Acute and chronic in vitro insulin treatment can inhibit the uptake of norepinephrine (NE) by adult rat brain synaptosomes and slices, fetal neuronal cultures, and PC12 cells. In the present study we tested whether chronic in vivo insulin treatment could alter the biosynthetic capacity of rat locus coeruleus neurons for the NE transporter protein (NET). Chronic third ventricular insulin treatment resulted in a suppression of NET mRNA to about one third of the level of vehicle-treated controls. Our finding suggests that insulin may play a regulatory role in the synthesis of this transporter, thereby modulating activity in CNS noradrenergic pathways.

Function and distribution of three rat 5-hydroxytryptamine7 (5-HT7) receptor isoforms produced by alternative splicing

Serotonin (5-HT7) receptor pre-mRNA is alternatively spliced in rat tissue to produce three isoforms, 5-HT(7a), 5-HT(7b) and 5-HT(7c), which differ in the amino acid sequences of their carboxyl terminal tails. Substantial species differences in structure and expression patterns exist for 5-HT7 isoforms. We have now compared some of the functional characteristics and level of expression for the three rat 5-HT7 receptor isoforms. Recombinant receptor isoforms were expressed in COS-7 cells for examination of [3H]5-HT binding characteristics and in JEG-3 cells to ascertain their ability to stimulate cAMP production. These studies showed that all three isoforms are functionally active and have similar agonist binding characteristics. Distribution of expression of the three rat receptor isoforms were examined in several brain regions and peripheral tissues using RT-PCR and in situ hybridization. The relative proportions of total 5-HT7 receptor message lent by each isoform varied little between these areas. In contrast to what has been observed in human tissue, the 5-HT(7a) isoform predominated in all regions examined, while the 5-HT(7c) isoform revealed a low level of expression (3% of total transcript). In situ hybridization was used to determine if the overall low level of expression of the 5-HT(7c) isoform by RT-PCR could be attributed to a small localized subpopulation of cells expressing high levels 5-HT(7c) message. In situ hybridization results indicate a generalized low level of expression of the 5-HT(7c) isoform throughout the CNS. These data suggest that while all three known 5-HT7 receptor isoforms in the rat are functionally competent, any functionally important differences between the three isoforms are not likely to involve differences in ligand binding or gross differences in adenylate cyclase coupling. However, differences in receptor phosphorylation, regulation or coupling to other effectors or cell trafficking could still exist.

Vasopressin in the septal area of the golden hamster controls scent marking and grooming

Microinjection of arginine vasopressin into the lateral septum and bed nucleus of the stria terminalis of male hamsters stimulates intense flank marking and flank gland grooming, while microinjections of vasopressin in sites immediately adjacent to these areas or in the lateral ventricle are ineffective. Microinjections of oxytocin, angiotensin II and the behaviorally active C-terminal fragment of vasopressin, metabolite neuropeptide, by comparison, do not stimulate flank marking. Effective sites for vasopressin injection are clearly superimposable upon autoradiographically defined sites of high V1-receptor density. Furthermore, vasopressin-sensitive neurons in the lateral septum and bed nucleus of the stria terminalis are necessary for the expression of naturally elicited flank marking since the microinjection of a V1-receptor antagonist into these sites was able to temporarily block flank marking triggered by odors from conspecifics.

Distribution of messenger RNA for the vasopressin V1a receptor in the CNS of male and female rats

The distribution of cells expressing mRNA encoding a vasopressin V1a receptor (V1aR) was examined in Long-Evans male and female rats by in situ hybridization using a [35S]cRNA probe. Specific hybridization to the vasopressin V1aR mRNA was evident in cells of the frontal cortex, piriform cortex, internal granular layer and the medial, dorsal, ventral and lateral portion of the anterior olfactory nucleus, zona limitans of the islands of Calleja, suprachiasmatic nucleus, CA1, CA2, CA3 and dentate gyrus of the hippocampus, paraventricular hypothalamic nucleus, ventromedial hypothalamic nucleus, arcuate nucleus, lateral habenular nucleus, and the molecular and granular cell layers of the cerebellum. The cerebellum, olfactory nucleus and the dentate gyrus appeared to be the most intensely labeled areas, while all other areas exhibited a lower level of expression. The anatomical distribution and the amount (as measured by optical density) of V1aR mRNA labeling was identical between male and female rats. This indicates that unlike the vasopressin gene itself, the expression of the vasopressin V1aR mRNA does not exhibit sexual dimorphism. These data demonstrate a wide spread distribution in the expression of the vasopressin V1aR mRNA in the CNS of male and female rats. This information on the anatomical distribution of the V1aR mRNA when combined with data concerning the anatomical distribution of the V1a binding sites, provides new information on the possible pre- and post-synaptic location of these neuropeptide receptors.

Diabetes causes differential changes in CNS noradrenergic and dopaminergic neurons in the rat: a molecular study

We have previously reported that chronic elevation of insulin in the CNS of rats results in opposing changes of the mRNA expression for the norepinephrine transporter (NET; decreased) and the dopamine transporter (DAT; increased). In the present study we tested the hypothesis that a chronic depletion of insulin would result in opposite changes of NET and DAT mRNA expression, from those observed with chronic elevation of insulin. Rats were treated with streptozotocin to produce hypoinsulinemic diabetes. One week later, steady state levels of mRNA were measured by in situ hybridization for NET in the locus coeruleus (LC) and for DAT in the ventral tegmental area/substantia nigra compacta (VTA/SNc). The mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme for NE and DA synthesis, was measured in these same brain regions. In the diabetic animals, NET mRNA was significantly elevated (159 +/- 22% of average control level) while DAT mRNA was non-significantly decreased (78 +/- 9% of average control level). Additionally, TH mRNA was significantly altered in both the LC (131 +/- 11% of average control level) and VTA/SNc (79 +/- 5% of average control level). We conclude that endogenous insulin is one physiological regulator of the synthesis and re-uptake of NE and DA in the CNS.

DEVELOPMENTAL SEIZURE SUSCEPTIBILITY OF KV1.1 POTASSIUM CHANNEL KNOCKOUT MICE

Potassium channels play a critical role in limiting neuronal excitability. Mutations in certain voltage-gated potassium channels have been associated with hyperexcitable phenotypes in both humans and animals. However, only recently have mutations in potassium channel genes (i.e. KCNQ2 and KCNQ3) been discovered in a human epilepsy, benign familial neonatal convulsions. Recently, it has been reported that mice lacking the voltage-gated Shaker-like potassium channel Kv1.1 α-subunit develop recurrent spontaneous seizures early in postnatal development. The clinical relevance of the Kv1.1 knockout mouse has been underscored by a recent report of epilepsy occurring in a family affected by mutations in the KCNA1 locus (the human homologue of Kv1.1) which typically cause episodic ataxia and myokymia. Here we summarize preliminary studies characterizing the developmental changes in seizure susceptibility and neuronal activation in the three genotypes of Kv1.1 mice (–/–, +/–, +/+). Using behavioral and immediate-early gene indicators of regional brain excitability, we have found that a seizure-sensitive predisposition exists in Kv1.1 –/– animals at a very young age (P10), before either spontaneous seizure activity or changes in c-fos mRNA expression can be demonstrated. Kv1.1 +/– mice, although behaviorally indistinguishable from wild types, also have an increased susceptibility to seizures at a similar early age. The Kv1.1 knockout mouse possesses many features desirable in a developmental animal epilepsy model and represents a clinically relevant model of early-onset epilepsies.