Modulation of cue-induced firing of ventral tegmental area dopamine neurons by leptin and ghrelin.

BACKGROUND/OBJECTIVES: The rewarding value of palatable foods contributes to overconsumption, even in satiated subjects. Midbrain dopaminergic activity in response to reward-predicting environmental stimuli drives reward-seeking and motivated behavior for food rewards. This mesolimbic dopamine (DA) system is sensitive to changes in energy balance, yet it has thus far not been established whether reward signaling of DA neurons in vivo is under control of hormones that signal appetite and energy balance such as ghrelin and leptin. SUBJECTS/METHODS: We trained rats (n=11) on an operant task in which they could earn two different food rewards. We then implanted recording electrodes in the ventral tegmental area (VTA), and recorded from DA neurons during behavior. Subsequently, we assessed the effects of mild food restriction and pretreatment with the adipose tissue-derived anorexigenic hormone leptin or the orexigenic hormone ghrelin on VTA DA reward signaling. RESULTS: Animals showed an increase in performance following mild food restriction (P=0.002). Importantly, food-cue induced DA firing increased when animals were food restricted (P=0.02), but was significantly attenuated after leptin pretreatment (P=0.00). While ghrelin did affect baseline DA activity (P=0.025), it did not affect cue-induced firing (P⩾0.353). CONCLUSIONS: Metabolic signals, such as leptin, affect food seeking, a process that is dependent on the formation of cue-reward outcomes and involves midbrain DA signaling. These data show that food restriction engages the encoding of food cues by VTA DA neurons at a millisecond level and leptin suppresses this activity. This suggests that leptin is a key in linking metabolic information to reward signaling.

The vital role of constitutive GPCR activity in the mesolimbic dopamine system.

The midbrain dopamine system has an important role in processing rewards and the stimuli associated with them, and is implicated in various psychiatric disorders. This system is tightly regulated by various G protein-coupled receptors (GPCRs). It is becoming increasingly clear that these receptors are not only activated by (endogenous) agonists but that they also exhibit agonist-independent intrinsic constitutive activity. In this review we highlight the evidence for the physiological role of such constitutive GPCR activity (in particular for cannabinoid 1, serotonin 2C and mu-opioid receptors) in the ventral tegmental area and in its output regions like the nucleus accumbens. We also address the behavioral relevance of constitutive GPCR signaling and discuss the repercussions of its abolition in dopamine-related psychiatric diseases.

Social isolation stress reduces hippocampal long-term potentiation: effect of animal strain and involvement of glucocorticoid receptors.

BACKGROUND: Depressive patients show cognitive impairments that are strongly associated with cortisol levels and hippocampus functioning that interact via unknown mechanisms. In addition, a relation between depression and hippocampal synaptic plasticity was described. METHODS: In the first experiment, strain-dependent effects of 72-h social isolation on long-term potentiation (LTP) in the CA1 area of the in vitro hippocampus, was determined. Extracellular field excitatory postsynaptic potentials were recorded and a brief high-frequency stimulation (100 Hz, 1s) was applied and recording resumed after the high frequency stimulation (HFS) for 30 min to determine the effect of HFS. In the second experiment we investigated the effect of 72 h of corticosterone treatment and the involvement of glucocorticoid receptors (GRs) in the effect of 72 h of social isolation on LTP in the CA1 area of hippocampus, in vitro. RESULTS: Genetic background has a major effect on the level of hippocampal LTP impairment in mice following social isolation. Data showed that the potentiation levels in socially housed (SH) A/J mice were significantly higher than the SH C57BL/6J mice (224.88 ± 16.65, 131.56 ± 6.25% of the baseline values, t(9)=2.648, p=0.026). However, both strains showed depressed induction of potentiation when reared in an isolated environment for 72 h, and no significant difference was recorded between the two (112.88 ± 16.65%, and 117.91 ± 3.23% of the baseline values, respectively, t(10)=0.618, p=0.551). Social isolation increased corticosterone levels significantly and chronic corticosterone infusion in SH phenocopied the LTP impairments observed in socially isolated mice. Infusion of the GR antagonist RU38486 rescued the LTP-impairments following social isolation. CONCLUSIONS: These findings support the notion that increased levels of stress hormone act via the GR on hippocampal functioning and that, in this way, the cognitive deficits in mood disorders may be restored.

Neutral antagonism at the cannabinoid 1 receptor: a safer treatment for obesity.

Obesity is a global problem with often strong neurobiological underpinnings. The cannabinoid 1 receptor (CB1R) was put forward as a promising drug target for antiobesity medication. However, the first marketed CB1R antagonist/inverse agonist rimonabant was discontinued, as its use was occasionally associated with negative affect and suicidality. In artificial cell systems, CB1Rs can become constitutively active in the absence of ligands. Here, we show that such constitutive CB1R activity also regulates GABAergic and glutamatergic neurotransmission in the ventral tegmental area and basolateral amygdala, regions which regulate motivation and emotions. We show that CB1R inverse agonists like rimonabant suppress the constitutive CB1R activity in such regions, and cause anxiety and reduced motivation for reward. The neutral CB1R antagonist NESS0327 does not suppress constitutive activity and lacks these negative effects. Importantly, however, both rimonabant and NESS0327 equally reduce weight gain and food intake. Together, these findings suggest that neutral CB1R antagonists can treat obesity efficiently and more safely than inverse agonists.

Presynaptic metabotropic glutamate receptors regulate glutamatergic input to dopamine neurons in the ventral tegmental area.

The ventral tegmental area is part of the midbrain dopamine system and is crucially involved in reward, motivation and drug abuse. The activity of dopamine neurons within this region is controlled by synaptic input. In particular, excitatory glutamatergic inputs are important for the switch from regular firing into burst firing. In the present manuscript we determined the role of presynaptic metabotropic glutamate receptors (mGluRs) in the regulation of spontaneous glutamate release of terminals projecting to dopamine cells in the ventral tegmental area of mice. We show that group III mGluRs regulate spontaneous glutamate release and this effect is most likely mediated by mGluR7. The presynaptic dampening of glutamatergic input might open new perspectives in the treatment of drug addiction.

Characterization of febrile seizures and febrile seizure susceptibility in mouse inbred strains.

Febrile seizures (FS) are the most prevalent seizures in children. Although FS are largely benign, complex FS increase the risk to develop temporal lobe epilepsy (TLE). Studies in rat models for FS have provided information about functional changes in the hippocampus after complex FS. However, our knowledge about the genes and pathways involved in the causes and consequences of FS is still limited. To enable molecular, genetic and knockout studies, we developed and characterized an FS model in mice and used it as a phenotypic screen to analyze FS susceptibility. Hyperthermia was induced by warm air in 10- to 14-day-old mice and induced FS in all animals. Under the conditions used, seizure-induced behavior in mice and rats was similar. In adulthood, treated mice showed increased hippocampal Ih current and seizure susceptibility, characteristics also seen after FS in rats. Of the seven genetically diverse mouse strains screened for FS susceptibility, C57BL/6J mice were among the most susceptible, whereas A/J mice were among the most resistant. Strains genetically similar to C57BL/6J also showed a susceptible phenotype. Our phenotypic data suggest that complex genetics underlie FS susceptibility and show that the C57BL/6J strain is highly susceptible to FS. As this strain has been described as resistant to convulsants, our data indicate that susceptibility genes for FS and convulsants are distinct. Insight into the mechanisms underlying seizure susceptibility and FS may help to identify markers for the early diagnosis of children at risk for complex FS and TLE and may provide new leads for treatment.

Prenatal corticosteroid exposure affects hippocampal plasticity and reduces lifespan.

Synthetic corticosteroids, such as dexamethasone, are frequently administered to pregnant women at risk for preterm delivery. Endogenous corticosteroids are essential for normal development, but exposure to therapeutic doses at critical developmental stages may have adverse effects on the central nervous system. Major concern has arisen about long-term effects of corticosteroid treatment on brain plasticity, particularly in the hippocampus. Therefore, we analyzed the molecular, cellular, and behavioral effects of prenatal dexamethasone treatment on the adult hippocampus. Pregnant mice were treated at embryonic day 15.5 with a single dose of dexamethasone or saline. Adult offspring was analyzed for hippocampal neuron loss, cell proliferation, and NMDA receptor subunit expression. Hippocampal function was assessed in the Morris water maze and synaptic plasticity in the CA1 field by determining frequency dependence of LTP and LTD in hippocampal slices. Prenatal dexamethasone treatment decreased hippocampal cell proliferation in the dentate gyrus. Treated mice showed reduced LTD, impaired spatial learning, and a marked reduction in lifespan. Our data show long-term adverse effects of prenatal dexamethasone treatment on hippocampal function in mice and suggest accelerated aging. These findings indicate that it is important to be restrictive with corticosteroid administration during fetal development because of the lifelong consequences.

Reduced psychostimulant effects on dopamine dynamics in the nucleus accumbens of mu-opioid receptor knockout mice.

Dopamine neurotransmission in the nucleus accumbens plays a pivotal role in the reinforcing properties of drugs of abuse. Two interacting processes regulate nucleus accumbens dopamine overflow: release of dopamine from presynaptic terminals and the subsequent reuptake by dopamine transporters. Opioid neurotransmission, primarily through mu-opioid receptors has also been strongly implicated in drug reward. We have previously shown that mice lacking the mu-opioid receptor display decreased cocaine self-administration. In addition, we found decreased impulse activity of midbrain dopaminergic neurons and an increased GABAergic input to these neurons in mu-opioid receptor knockout mice. In the present study we investigated whether these changes in dopaminergic cell bodies are accompanied by altered dopamine dynamics at the terminal level. To that aim, we measured nucleus accumbens dopamine overflow using fast scan cyclic voltammetry. Our data demonstrate that in mu-opioid receptor knockout mice 1) the reuptake of dopamine in the nucleus accumbens is slower, and 2) the relative effect of cocaine and amphetamine on the reuptake of dopamine is smaller compared with wild type mice. These data provide a mechanism for the decreased reinforcing properties of cocaine observed in mu-opioid receptor knockout mice.

Diabetes mellitus concomitantly facilitates the induction of long-term depression and inhibits that of long-term potentiation in hippocampus.

Memory impairments, which occur regularly across species as a result of ageing, disease (such as diabetes mellitus) and psychological insults, constitute a useful area for investigating the neurobiological basis of learning and memory. Previous studies in rats found that induction of diabetes (with streptozotocin, STZ) impairs long-term potentiation (LTP) but enhances long-term depression (LTD) induced by high- (HFS) and low-frequency stimulations (LFS), respectively. Using a pairing protocol under whole-cell recording conditions to induce synaptic plasticity at Schaffer collateral synapses in hippocampal CA1 slices, we show that LTD and LTP have similar magnitudes in diabetic and age-matched control rats. But, in diabetic animals, LTD is induced at more polarized and LTP more depolarized membrane potentials (V(ms)) compared with controls: diabetes produces a 10 mV leftward shift in the threshold for LTD induction and 10 mV rightward shift in the LTD-LTP crossover point of the voltage-response curve for synaptic plasticity. Prior repeated short-term potentiations or LTP are known to similarly, though reversibly, lower the threshold for LTD induction and raise that for LTP induction. Thus, diabetes- and activity-dependent modulation of synaptic plasticity (referred to as metaplasticity) display similar phenomenologies. In addition, compared with naïve synapses, prior induction of LTP produces a 10 mV leftward shift in Vms for inducing subsequent LTD in control but not in diabetic rats. This could indicate that diabetes acts on synaptic plasticity through mechanisms involved in metaplasticity. Persistent facilitation of LTD and inhibition of LTP may contribute to learning and memory impairments associated with diabetes mellitus.

Increased gabaergic input to ventral tegmental area dopaminergic neurons associated with decreased cocaine reinforcement in mu-opioid receptor knockout mice.

There is general agreement that dopaminergic neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens and prefrontal cortex play a key role in drug reinforcement. The activity of these neurons is strongly modulated by the inhibitory and excitatory input they receive. Activation of mu-opioid receptors, located on GABAergic neurons in the VTA, causes hyperpolarization of these GABAergic neurons, thereby causing a disinhibition of VTA dopaminergic neurons. This effect of mu-opioid receptors upon GABA neurotransmission is a likely mechanism for mu-opioid receptor modulation of drug reinforcement. We studied mu-opioid receptor signaling in relation to cocaine reinforcement in wild-type and mu-opioid receptor knockout mice using a cocaine self-administration paradigm and in vitro electrophysiology. Cocaine self-administration was reduced in mu-opioid receptor knockout mice, suggesting a critical role of mu-opioid receptors in cocaine reinforcement. The frequency of spontaneous inhibitory post-synaptic currents onto dopaminergic neurons in the ventral tegmental area was increased in mu-opioid receptor knockout mice compared with wild-type controls, while the frequency of spontaneous excitatory post-synaptic currents was unaltered. The reduced cocaine self-administration and increased GABAergic input to VTA dopaminergic neurons in mu-opioid receptor knockout mice supports the notion that suppression of GABAergic input onto dopaminergic neurons in the VTA contributes to mu-opioid receptor modulation of cocaine reinforcement.

Increased spike broadening and slow afterhyperpolarization in CA1 pyramidal cells of streptozotocin-induced diabetic rats.

Diabetes mellitus is associated with impairments of cognitive function both in humans and animal models. In diabetic rats cognitive deficits are related to alterations in activity-dependent synaptic plasticity in the hippocampus. Many similarities with the pathophysiology of normal brain aging have been noted, and the view emerges that the effects of diabetes on the brain are best described as "accelerated brain aging."In the present study we examined whether CA1 pyramidal neurons from streptozotocin-induced diabetic rats display an increased slow afterhyperpolarization, often considered as a hallmark of neuronal aging. We found no differences in resting membrane potential, input resistance, membrane time-constant, and action potential amplitude and duration between CA1 pyramidal neurons from streptozotocin-induced diabetic and age-matched control rats. During a train of action potentials, however, there is an increased broadening of the action potentials in diabetic animals, so-called "spike broadening." The amplitude of the slow afterhyperpolarization elicited by a train of action potentials is indeed increased in diabetic animals. Interestingly, when the slow afterhyperpolarization is elicited by a Ca(2+) spike, there is no difference between control and diabetic rats. This indicates that the increased slow afterhyperpolarization in diabetes is likely to be due to an increased Ca(2+) influx resulting from the increased spike broadening. These data underscore the notion that the diabetic brain at the neuronal level shares properties with brain aging.

Effects of streptozotocin-diabetes on the hippocampal NMDA receptor complex in rats.

In animal models of diabetes mellitus, such as the streptozotocin-diabetic rat (STZ-rat), spatial learning impairments develop in parallel with a reduced expression of long-term potentiation (LTP) and enhanced expression of long-term depression (LTD) in the hippocampus. This study examined the time course of the effects of STZ-diabetes and insulin treatment on the hippocampal post-synaptic glutamate N-methyl-D-aspartate (NMDA) receptor complex and other key proteins regulating hippocampal synaptic transmission in the post-synaptic density (PSD) fraction. In addition, the functional properties of the NMDA-receptor complex were examined. One month of STZ-diabetes did not affect the NMDA receptor complex. In contrast, 4 months after induction of diabetes NR2B subunit immunoreactivity, CaMKII and Tyr-dependent phosphorylation of the NR2A/B subunits of the NMDA receptor were reduced and alphaCaMKII autophosphorylation and its association to the NMDA receptor complex were impaired in STZ-rats compared with age-matched controls. Likewise, NMDA currents in hippocampal pyramidal neurones measured by intracellular recording were reduced in STZ-rats. Insulin treatment prevented the reduction in kinase activities, NR2B expression levels, CaMKII-NMDA receptor association and NMDA currents. These findings strengthen the hypothesis that altered post-synaptic glutamatergic transmission is related to deficits in learning and plasticity in this animal model.

Interaction between synaptic excitation and slow afterhyperpolarization current in rat hippocampal pyramidal cells.

1. Whole cell recordings from CA1 pyramidal cells were performed to investigate the interaction between excitatory postsynaptic potentials (EPSPs) or currents (EPSCs), and the slow Ca(2+)-dependent K(+) current, I(sAHP). Blockers of the slow afterhyperpolarization (sAHP) such as isoprenaline (ISO) or noradrenaline (NA) reduced the hyperpolarization that followed a short train of EPSPs, and slowed the decay of summated EPSPs or EPSCs. 2. ISO/NA action on synaptic responses was observed in the absence of action potentials, but was curtailed by Ca(2+) chelation (10 mM EGTA in the electrode) and was not observed with a caesium-based recording solution. This suggests the involvement of an ISO/NA-sensitive Ca(2+)-dependent K(+) current without a requirement for regenerative spiking. 3. An ISO/NA-sensitive sAHP was observed following both NMDA and non-NMDA receptor-mediated EPSP trains in nominally zero Mg(2+) medium. Isoprenaline sensitivity was blocked by hyperpolarization during EPSPs or by isradipine, suggesting a requirement for voltage-dependent Ca(2+) influx during EPSPs. The data indicate that bursts of EPSPs can activate voltage-gated Ca(2+) channels, which trigger I(sAHP) during synaptic responses. 4. A decrease in EPSP temporal summation occurred during both spike-evoked sAHPs and persistent activation of sAHP conductance following internal dialysis with diazo-2 (2 mM). At constant membrane potential, diazo-2 caused a decrease in membrane time constant and input resistance and accelerated the rate of EPSP decay. Photolysis of diazo-2 or application of NA reduced the resting sAHP conductance, causing an increased membrane time constant and input resistance in association with an increase in EPSP half-width. 5. These results indicate that short bursts of EPSPs can activate a Ca(2+)-dependent K(+) current resembling I(sAHP), and that activation of this current reduces the postsynaptic response to high-frequency synaptic input. The findings imply that modulation of I(sAHP) can regulate synaptic efficacy and may influence the threshold for tetanus-induced synaptic plasticity.

Long term depression in the CA1 field is associated with a transient decrease in pre- and postsynaptic PKC substrate phosphorylation.

Induction of homosynaptic long term depression (LTD) in the CA1 field of the hippocampus is thought to require activation of N-methyl-d-aspartate receptors, an elevation of postsynaptic Ca(2+) levels, and a subsequent increase in phosphatase activity. To investigate the spatial and temporal changes in protein phosphatase activity following LTD induction, we determined the in situ phosphorylation state of a pre- (GAP-43/B-50) and postsynaptic (RC3) protein kinase C substrate during N-methyl-d-aspartate receptor-dependent LTD in the CA1 field of rat hippocampal slices. We show that LTD is associated with a transient (<30 min) and D-AP5-sensitive reduction in GAP-43/B-50 and RC3 phosphorylation and that LTD is prevented by the phosphatase inhibitors okadaic acid and cyclosporin A. Our data provide strong evidence for a transient increase in pre- and postsynaptic phosphatase activity during LTD. Since the in situ phosphorylation of the calmodulin-binding proteins GAP-43/B-50 and RC3 changes during both LTD and long term potentiation, these proteins may form part of the link between the Ca(2+) signal and Ca(2+)/calmodulin-dependent processes implicated in long term potentiation and LTD.

Activation of pre- and postsynaptic protein kinase C during tetraethylammonium-induced long-term potentiation in the CA1 field of the hippocampus.

Tetraethylammonium (TEA) induces a form of long-term potentiation (LTP) that is independent on N-methyl-D-aspartate (NMDA) receptor activation (LTP(K)). LTP(K) may be a suitable chemical model to study molecular mechanisms underlying LTP. We monitored the phosphorylation state of two identified neural-specific protein kinase C (PKC) substrates (the presynaptic protein GAP-43/B-50 and postsynaptic protein RC3) after different chemical depolarisations. TEA induced a long-lasting increase in synaptic efficacy in the CA1 field of the hippocampus and increased the phosphorylation of both GAP-43/B-50 and RC3 (51 and 56.1%, respectively). These effects were blocked by the voltage-dependent calcium channel antagonist nifedipine, but not by the NMDA receptor antagonist AP5. These data show that in LTP(K) the in situ phosphorylation of pre-and postsynaptic PKC substrates is increased, indicating that NMDA receptor-dependent and NMDA receptor-independent LTP share common Ca(2+)-dependent expression mechanisms, including activation of pre- and postsynaptic PKC.

Chemical LTD in the CA1 field of the hippocampus from young and mature rats.

Within the hippocampal formation, two forms of long-lasting synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD), can be induced which require the activation of NMDA receptors. Interestingly, it has been shown that both LTP and LTD are reduced in adult animals. Recently, a new chemical protocol has been described which elicits LTD in the CA1 field of the hippocampus. Application of 20 microM NMDA for 3 min results in a stable and long-lasting decrease in the evoked synaptic responses. We used this protocol to induce LTD in hippocampal slices from young and adult rats and show that this form of LTD is AP5-sensitive and can be blocked by the protein phosphatase inhibitor cyclosporin A in slices from adult animals. In contrast to electrical LTD (induced by prolonged low frequency stimulation), the extent of chemical LTD was not different between the young and adult rats. These findings indicate that the intracellular signal transduction cascades involved in long-lasting synaptic depression are still intact in adult animals.

Differential changes in the phosphorylation of the protein kinase C substrates myristoylated alanine-rich C kinase substrate and growth-associated protein-43/B-50 following Schaffer collateral long-term potentiation and long-term depression.

Activation of protein kinase C (PKC) is one of the biochemical pathways thought to be activated during activity-dependent synaptic plasticity in the brain, and long-term potentiation (LTP) and long-term depression (LTD) are two of the most extensively studied models of synaptic plasticity. Here we have examined changes in the in situ phosphorylation level of two major PKC substrates, myristoylated alanine-rich C kinase substrate (MARCKS) and growth-associated protein (GAP)-43/B-50, after pharmacological stimulation or induction of LTP or LTD in the CA1 field of the hippocampus. We find that direct PKC activation with phorbol esters, K+-induced depolarization, and activation of metabotropic glutamate receptors increase the in situ phosphorylation of both MARCKS and GAP-43/B-50. The induction of LTP increased the in situ phosphorylation of both MARCKS and GAP-43/B-50 at 10 min following high-frequency stimulation, but only GAP-43/B-50 phosphorylation remained elevated 60 min after LTP induction. Furthermore, blockade of LTP induction with the NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid prevented elevations in GAP-43/B-50 phosphorylation but did not prevent the elevation in MARCKS phosphorylation 10 min following LTP induction. The induction of LTD resulted in a reduction in GAP-43/B-50 phosphorylation but did not affect MARCKS phosphorylation. Together these findings show that activity-dependent synaptic plasticity elicits PKC-mediated phosphorylation of substrate proteins in a highly selective and coordinated manner and demonstrate the compartmentalization of PKC-substrate interactions. Key Words: Protein kinase C-Myristoylated alanine-rich C kinase substrate-Growth-associated protein-43-Long-term potentiation-Long-term depression-(RS)-alpha-Methyl-4-carboxyphenylglycine-D-2-Amino-5-ph osphonopentanoic acid-Glutamate.

Substrate phosphorylation in the protein kinase Cgamma knockout mouse.

The phosphorylation state of three identified neural-specific protein kinase C substrates (RC3, GAP-43/B-50, and MARCKS) was monitored in hippocampal slices of mice lacking the gamma-subtype of protein kinase C and wild-type controls by quantitative immunoprecipitation following 32Pi labeling. Depolarization with potassium, activation of glutamate receptors with glutamate, or direct stimulation of protein kinase C with a phorbol ester increased RC3 phosphorylation in wild-type animals but failed to affect RC3 phosphorylation in mice lacking the gamma-subtype of protein kinase C. Our results suggests the following biochemical pathway: activation of a postsynaptic (metabotropic) glutamate receptor stimulates the gamma-subtype of protein kinase C, which in turn phosphorylates RC3. The inability to increase RC3 phosphorylation in mice lacking the gamma-subtype of protein kinase C by membrane depolarization or glutamate receptor activation may contribute to the spatial learning deficits and impaired hippocampal LTP observed in these mice.

Protein kinase C in synaptic plasticity: changes in the in situ phosphorylation state of identified pre- and postsynaptic substrates.

1. Long-term potentiation and its counterpart long-term depression are two forms of activity dependent synaptic plasticity, in which protein kinases and protein phosphatases are essential. 2. B-50/GAP-43 and RC3/neurogranin are two defined neuronal PKC substrates with different synaptic localization. B-50/GAP-43 is a presynaptic protein and RC3/neurogranin is only found at the postsynaptic site. Measuring their phosphorylation state in hippocampal slices, allows us to simultaneously monitor changes in pre- and postsynaptic PKC mediated phosphorylation. 3. Induction of LTP in the CA1 field of the hippocampus is accompanied with an increase in the in situ phosphorylation of both B-50/GAP-43 and RC3/neurogranin, during narrow, partially overlapping, time windows. 4. Pharmacological data show that mGluR stimulation results in an increase in the in situ phosphorylation of B-50/GAP-43 and RC3/neurogranin.