Joseph B. Watson

Effects of repeated amphetamine treatment on the locomotor activity of the dopamine D1A-deficient mouse

THE role of dopamine D1A receptors in mediating amphetamine-induced sensitization was investigated using the D1A-deficient mouse. During the drug pre-exposure phase, D1A-deficient and control mice were injected for five consecutive days with saline or amphetamine (2 mg/kg, i.p.). Locomotor activity was measured on the first and fifth pre-exposure day. After three abstinence days, mice were given either amphetamine or saline and locomotor activity was again assessed. Mice were then sacrificed and protein kinase A (PKA) activity was measured. In contrast to control mice, D1A-deficient mice did not show a progressive increase in locomotor activity across days. Importantly, both control and mutant mice did exhibit behavioral sensitization, because mice pre-exposed and tested with amphetamine were more active than mice acutely tested with the drug. Even so, the amphetamine-induced locomotor activity of the mutant mice was significantly reduced when compared with similarly treated control mice, indicating that the sensitized response was less pronounced in the D1A-deficient mouse. PKA activity also varied depending on genotype, since amphetamine decreased PKA activity in control but not D1A-deficient mice.

Effects of repeated methylphenidate treatment in the young rat: sensitization of both locomotor activity and stereotyped sniffing.

The behavioral effects of repeated methylphenidate (MPH) treatment were assessed in young rats. In 4 experiments, rats (starting at Postnatal Day 10 or 16) were pretreated on 5 consecutive days with saline or MPH (2.5-20.0 mg/kg i.p.). Sensitization was assessed after 1 or 7 abstinence days, with rats receiving a test day challenge injection of either a low dose of MPH (2.5 mg/kg) or the same dose of MPH as given during pretreatment. Results show that a test day injection of 2.5 mg/kg MPH produced a sensitized locomotor response in rats pretreated with 2.5-20.0 mg/kg MPH. This MPH-induced locomotor sensitization was evident only after 1 abstinence day. Various pretreatment doses of MPH (5, 10, 15, or 20 mg/kg) were capable of sensitizing the stereotyped sniffing of young rats, but only rats pretreated and tested with the highest dose (20 mg/kg) of MPH showed an augmented stereotyped sniffing response that was still robust after 7 abstinence days. Results indicate that young rats are capable of exhibiting sensitization after an extended abstinence period, which contrasts with previous research suggesting that psychostimulant treatment does not produce long-term sensitization in young rats.

Adenylyl cyclase-dependent form of chemical long-term potentiation triggers translational regulation at the elongation step.

The persistent maintenance of long-term potentiation requires both messenger RNA and protein synthesis. While there is mounting evidence for an active role of protein synthesis in hippocampal long-term potentiation, the nature of mechanisms underlying its regulation has not yet been established. We used a previously described chemical long-term potentiation protocol [J Neurosci 19 (1999) 2500] to address the hypothesis that signaling mechanisms, involved in long-lasting long-term potentiation, directly regulate protein synthesis. Chemical long-term potentiation is an N-methyl-D-aspartate receptor-dependent form of plasticity, which relies on both synaptic activity, in the form of spontaneous bursting induced by high concentrations of K(+) and Ca(2+), and cyclic AMP/adenylyl cyclase signaling. We found that chemical long-term potentiation in CA1 of the mouse hippocampus lasts for at least 3 hours and requires both messenger RNA and protein synthesis. However, surprisingly de novo total protein synthesis was paradoxically decreased at 1 hour after long-term potentiation induction. Consistent with the decrease in total protein synthesis in potentiated CA1, phosphorylation of eukaryotic elongation factor 2 was increased and is likely responsible for inhibition of translation at the elongation step. Increased phosphorylation of eukaryotic elongation factor 2 was dependent on coincident cyclic AMP/adenylyl cyclase activation and synaptic activity and required N-methyl-D-aspartate receptor activation. Despite the inhibition in total protein synthesis, the level of the immediate early gene protein Arc (activity regulated cytoskeleton-associated protein) increased at 1 hour after chemical long-term potentiation induction. Taken together, the results suggest that regulation at the elongation step of protein synthesis contributes to persistent forms of long-term potentiation.

Alterations in corticostriatal synaptic plasticity in mice overexpressing human α-synuclein

Most forms of Parkinsons disease (PD) are sporadic in nature, but some have genetic causes as first described for the alpha-synuclein gene. The alpha-synuclein protein also accumulates as insoluble aggregates in Lewy bodies in sporadic PD as well as in most inherited forms of PD. The focus of the present study is the modulation of synaptic plasticity in the corticostriatal pathway of transgenic (Tg) mice that overexpress the human alpha-synuclein protein throughout the brain (ASOTg). Paired-pulse facilitation was detected in vitro by activation of corticostriatal afferents in ASOTg mice, consistent with a presynaptic effect of elevated human alpha-synuclein. However basal synaptic transmission was unchanged in ASOTg, suggesting that human alpha-synuclein could impact paired-pulse facilitation via a presynaptic mechanism not directly related to the probability of neurotransmitter release. Mice lacking alpha-synuclein or those expressing normal and A53T human alpha-synuclein in tyrosine hydroxylase-containing neurons showed, instead, paired-pulse depression. High-frequency stimulation induced a presynaptic form of long-term depression solely in ASOTg striatum. A presynaptic, N-methyl-d-aspartate receptor-independent form of chemical long-term potentiation induced by forskolin (FSK) was enhanced in ASOTg striatum, while FSK-induced cAMP levels were reduced in ASOTg synaptoneurosome fractions. Overall the results suggest that elevated human alpha-synuclein alters presynaptic plasticity in the corticostriatal pathway, possibly reflecting a reduction in glutamate at corticostriatal synapses by modulation of adenylyl cyclase signaling pathways. ASOTg mice may recapitulate an early stage in PD during which overexpressed alpha-synuclein dampens corticostriatal synaptic transmission and reduces movement.

Dendritic Translocation of RC3/Neurogranin mRNA in Normal Aging, Alzheimer Disease and Fronto-Temporal Dementia

RC3/neurogranin is a postsynaptic protein kinase C (PKC)-/calmodulin-binding substrate implicated in long-term potentiation (LTP) forms of synaptic plasticity. Our previous digoxigenin in situ hybridization (DIG-ISH) studies detected RC3 mRNA in apical dendrites and cell bodies of neurons in the rat cerebral cortex and hippocampus. This observation suggested that RC3 mRNA is selectively translocated to dendrites, where it may be translated locally in response to synaptic activity. To test this hypothesis further, we isolated a full-length cDNA clone of the homologous human RC3 mRNA from a human cortex γGT11 library, determined its nucleotide and predicted amino acid sequences, and performed mRNA expression studies in cerebral cortex from normal human patients and from patients with Alzheimer disease (AD) and fronto-temporal dementia (FTD). The human cDNA clone detects a single ≈1.3 kb mRNA whose nucleotide sequence is 73% similar to the rat nucleotide sequence and 96% similar to its amino acid sequence. DIG-ISH studies detect robust staining of RC3 mRNA in cell bodies of numerous neurons throughout Layers II-VI and in both apical and basal dendrites of pyramidal neurons in human neocortex (temporal/frontal). We conclude that dendritic targeting of RC3 mRNA is conserved in human brain. In AD neocortex tissue, there is little or no evidence for RC3 mRNA translocation to dendrites, while in FTD neocortex, targeting of RC3 mRNA to apical dendrites is preserved. Comparative studies in AD and FTD point to the potential importance of synapse integrity and the dendritic cytoskeleton in RC3 mRNA targeting in the human neocortex.

Multiple Sources of Striatal Inhibition Are Differentially Affected in Huntington's Disease Mouse Models

In Huntingtons disease (HD) mouse models, spontaneous inhibitory synaptic activity is enhanced in a subpopulation of medium-sized spiny neurons (MSNs), which could dampen striatal output. We examined the potential source(s) of increased inhibition using electrophysiological and optogenetic methods to assess feedback and feedforward inhibition in two transgenic mouse models of HD. Single whole-cell patch-clamp recordings demonstrated that increased GABA synaptic activity impinges principally on indirect pathway MSNs. Dual patch recordings between MSNs demonstrated reduced connectivity between MSNs in HD mice. However, while connectivity was strictly unidirectional in controls, in HD mice bidirectional connectivity occurred. Other sources of increased GABA activity in MSNs also were identified. Dual patch recordings from fast spiking (FS) interneuron–MSN pairs demonstrated greater but variable amplitude responses in MSNs. In agreement, selective optogenetic stimulation of parvalbumin-expressing, FS interneurons induced significantly larger amplitude MSN responses in HD compared with control mice. While there were no differences in responses of MSNs evoked by activating single persistent low-threshold spiking (PLTS) interneurons in recorded pairs, these interneurons fired more action potentials in both HD models, providing another source for increased frequency of spontaneous GABA synaptic activity in MSNs. Selective optogenetic stimulation of somatostatin-expressing, PLTS interneurons did not reveal any significant differences in responses of MSNs in HD mice. These findings provide strong evidence that both feedforward and to a lesser extent feedback inhibition to MSNs in HD can potentially be sources for the increased GABA synaptic activity of indirect pathway MSNs.

Localization of RC3 (neurogranin) in rat brain subcellular fractions

Previous studies have shown that RC3 (neurogranin) is a postsynaptic, protein kinase C (PKC)/calmodulin-binding substrate that accumulates throughout the perikaryal and dendritic cytoplasm and is often closely associated with the postsynaptic density (PSD) in dendritic spines of neostriatal neurons. Here Western immunoblotting studies of rat brain subcellular fractions confirm that RC3 is predominantly a cytosolic protein but is found in lower amounts in membrane-enriched microsomes and synaptosomes. Solubilization of synaptosomes suggests that RC3 may only be loosely associated with the PSD.

Differential cDNA Screening Strategies to Identify Novel Stage-Specific Proteins in the Developing Mammalian Brain

The molecular basis for cell lineage, migration and patterning in the developing mammalian brain is the focus of extensive investigation by neuroscientists. Many of the proteins that are uniquely expressed in specific stages of development remain to be isolated. In the present review, subtractive hybridization, differential display and single cell amplification are presented as possible differential cDNA screening strategies to identify new proteins important to neural development. The advantages and limitations of each approach are discussed and new lines of investigation in which they can be applied effectively are suggested.

Functional consequences of expression of the neuron-specific, protein kinase C substrate RC3 (neurogranin) in Xenopus oocytes.

RC3 (neurogranin) is a neuron-specific substrate of protein kinase C (PKC) that accumulates predominantly in dendritic spines of forebrain neurons and undergoes long-term potentiation (LTP)-associated increases in PKC-phosphorylation in hippocampal slices. Here the hypothesis that RC3 functions by modulating the IP3/DAG second messenger pathway after its phosphorylation by DAG-activated PKC was tested by heterologous expression in Xenopus oocytes. Acetylcholine-evoked inward chloride (Cl-) currents, dependent on both IP3 release and intracellular calcium (Ca2+), were 2- to 3-fold higher in RC3-injected oocytes than in uninjected control oocytes. RC3-oocytes did not exhibit enhanced currents when preincubated with the protein kinase inhibitor H-7 or when a glycine residue was substituted for serine, the PKC phosphorylation site of RC3. Activation of endogenous oocyte PKC by phorbol esters generated inward Cl- currents in RC3 oocytes but not in control oocytes. RC3-dependent Cl- currents were also elicited by phorbol ester in Ca(2+)-free media. We propose that PKC-phosphorylated RC3 is capable of enhancing the mobilization of intracellular Ca2+ in Xenopus oocytes and, by inference, may play a role in Ca2+ homeostasis in dendrites of forebrain neurons.

Age‐related deficits in long‐term potentiation are insensitive to hydrogen peroxide: Coincidence with enhanced autophosphorylation of Ca2+/calmodulin‐dependent protein kinase II

Reactive oxygen species (ROS) can have deleterious effects for both normal aging and Alzheimers disease (AD). We examined the hypothesis that synapses undergoing long‐term potentiation (LTP) are preferentially at risk for ROS‐mediated oxidative stress during aging. We observed age‐dependent deficits in LTP induced by a high‐frequency stimulation (HFS) protocol in the CA1 region of hippocampus from C57BL/6 mice. There was a significant difference between LTP measured over 60 min in young (1–2 months) and old (23–26 months) mice. In oxidative stress studies, exogenous H2O2 (580 μM) significantly inhibited LTP in young mice; a similar dose of H2O2 failed to inhibit LTP in slices from adult (2–4 months) or from old mice. The results show that there are significant deficits in LTP in aging mice, but such deficits are insensitive to H2O2. Western immunoblotting studies in young mice show that the relative levels of autophosphorylated α‐Ca2+/calmodulin‐dependent protein kinase II (CaMKII) are unchanged in hippocampal CA1 treated with H2O2 relative to untreated controls. However with aging, there is a significant enhancement in the levels of autophosphorylated CaMKII in H2O2‐treated CA1 of older mice. Phosphorylation of RC3/neurogranin (Ng) by protein kinase C (PKC) is decreased in CA1 in response to H2O2 treatment, irrespective of age. We propose that, during aging, enhanced local release of H2O2 from mitocohondria may induce a compensatory “ceiling” effect at synapses, so that the levels of autophosphorylated αCaMKII are aberrantly saturated, leading to alterations in synaptic plasticity.