PROSPER delivery of universal preventive interventions with young adolescents: long-term effects on emerging adult substance misuse and associated risk behaviors.

BACKGROUND: Substance misuse and associated health-risking behaviors are prevalent in emerging adulthood. There is a knowledge gap concerning the post-high school effects of community-based delivery systems for universal preventive interventions implemented during young adolescence. This study reports effects of the PROSPER delivery system through age 19, 7.5 years past baseline. METHODS: A cohort sequential design included 28 public school districts randomly assigned to the PROSPER partnership delivery system or usual-programming conditions. PROSPER community teams implemented a family-focused intervention in 6th grade and a school-based intervention in 7th grade. Outcomes for the age 19, post-high school report included lifetime, current, and frequency of substance misuse, as well as antisocial and health-risking sexual behaviors. Intent-to-treat, multi-level analyses of covariance of point-in-time outcomes were conducted, along with analyses of risk-related moderation of intervention effects. RESULTS: Results showed emerging adults from PROSPER communities reported significantly lower substance misuse across a range of types of substances, with relative reduction rates of up to 41.0%. No significant findings were observed for associated antisocial and health-risking sexual behavior indices; or for lifetime rates of sexually transmitted infections. Risk-related moderation effects were non-significant, suggesting generally comparable outcomes across higher- and lower-risk subgroups of emerging adults. CONCLUSIONS: The PROSPER delivery system for brief universal preventive interventions has potential for public health impact by reducing long-term substance misuse, with positive results extending beyond high school.

Chronic activation of the D2 autoreceptor inhibits both glutamate and dopamine synapse formation and alters the intrinsic properties of mesencephalic dopamine neurons in vitro.

Dysfunctional dopamine (DA)-mediated signaling is implicated in several diseases including Parkinson's disease, schizophrenia and attention deficit and hyperactivity disorder. Chronic treatment with DA receptor agonists or antagonists is often used in pharmacotherapy, but the consequences of these treatments on DA neuron function are unclear. It was recently demonstrated that chronic D2 autoreceptor (D2R) activation in DA neurons decreases DA release and inhibits synapse formation. Given that DA neurons can establish synapses that release glutamate in addition to DA, we evaluated the synapse specificity of the functional and structural plasticity induced by chronic D2R activation. We show that chronic activation of the D2R with quinpirole in vitro caused a parallel decrease in the number of dopaminergic and glutamatergic axon terminals. The capacity of DA neurons to synthesize DA was not altered, as indicated by the lack of change in protein kinase A-mediated Ser(40) phosphorylation of tyrosine hydroxylase. However, the spontaneous firing rate of DA neurons was decreased and was associated with altered intrinsic properties as revealed by a prolonged latency to first spike after release from hyperpolarization. Moreover, D2R function was decreased after its chronic activation. Our results demonstrate that chronic activation of the D2R induces a complex neuronal reorganization involving the inhibition of both DA and glutamate synapse formation and an alteration in electrical activity, but not in DA synthesis. A better understanding of D2R-induced morphological and functional long-term plasticity may lead to improved pharmacotherapy of DA-related neurological and psychiatric disorders.

Critical roles for the netrin receptor deleted in colorectal cancer in dopaminergic neuronal precursor migration, axon guidance, and axon arborization.

DCC (deleted in colorectal cancer), a receptor for the axon guidance cue netrin-1, is highly expressed by mesencephalic dopaminergic (DA) neurons during development; however, the contribution of DCC to DA development remains largely uncharacterized. DA neurons in ventral mesencephalic nuclei also express UNC5 homologue netrin receptors from late embryogenesis to adulthood, raising the possibility that DA axons could be attracted or repelled by netrins. Examining newborn dcc null mice, we report that loss of DCC function results in profound alterations of DA circuitry, including DA progenitor cell migration defects, reduced numbers of DA cells in midbrain nuclei, an anomalous DA ventral commissure, malformed DA innervation of the ventral striatum, and reduced DA innervation of the cerebral cortex. Caspase-3 activation was detected in inappropriately localized DA cells, consistent with apoptosis contributing to reduced cell numbers. Dcc heterozygous mice express reduced levels of DCC protein. Although less severely disrupted than dcc nulls, newborn and adult dcc heterozygotes also have fewer DA neurons in ventral mesenscephalic nuclei. Despite the reduced numbers of DA neurons, newborn dcc heterozygotes and nulls exhibit similar DA innervation density as wild-type littermates in the nucleus accumbens core, and adult dcc heterozygotes exhibit increased DA innervation in medial prefrontal cortex. A trend towards increased innervation of medial prefrontal cortex was detected in newborn dcc heterozygotes, but did not reach statistical significance, suggesting that the increase in adult heterozygotes results from enhanced DA arborization during postnatal development. Consistent with the hypothesis that DCC regulates DA axonal projections, disrupting DCC function in culture inhibits netrin-1 induced DA axon extension and axon branching. Furthermore, disrupting DCC function in isolated DA neurons grown as micro-island cultures reduces the number of autaptic synapses per cell. We conclude that DCC regulates appropriate precursor cell migration, axon guidance, and terminal arborization by DA neurons.

Chronic activation of the D2 dopamine autoreceptor inhibits synaptogenesis in mesencephalic dopaminergic neurons in vitro.

Chronic blockade or activation of dopamine receptors is critical for the pharmacological treatment of diseases like schizophrenia, Parkinson's or attention deficit and hyperactivity disorder. However, the long-term impact of such treatments on dopamine neurons is unclear. Chronic blockade of the dopamine D2 receptor in vivo triggers an increase in the axonal arborization of dopamine neurons [European Journal of Neuroscience, 2002, 16, 787-794]. However, the specific involvement of presynaptic (autoreceptors) vs. postsynaptic D2 receptors as well as the molecular mechanisms involved have not been determined. Here, we examined the role of D2 autoreceptors in regulating the ability of mouse dopamine neurons to establish axon terminals. Chronic activation of this receptor with quinpirole, a specific agonist, decreased the number of axon terminals established by isolated dopamine neurons. This effect was accompanied by a decrease in dopamine release and was mediated through inhibition of protein kinase A. The decrease in axon terminal number induced by D2 receptor activation was also occluded when the mammalian Target of Rapamycin pathway of mRNA translation was blocked. Our results suggest that chronic activation of the D2 autoreceptor inhibits synaptogenesis by mesencephalic dopamine neurons through translational regulation of the synthesis of proteins required for synapse formation. This study provides a better understanding of the impact of long-term pharmacological interventions acting through the D2 receptor.

Enhanced glutamatergic phenotype of mesencephalic dopamine neurons after neonatal 6-hydroxydopamine lesion.

There is increasing evidence that a subset of midbrain dopamine (DA) neurons uses glutamate as a co-transmitter and expresses vesicular glutamate transporter (VGLUT) 2, one of the three vesicular glutamate transporters. In the present study, double in situ hybridization was used to examine tyrosine hydroxylase (TH) and VGLUT2 mRNA expression during the embryonic development of these neurons, and postnatally, in normal rats and rats injected with 6-hydroxydopamine (6-OHDA) at P4 to destroy partially DA neurons. At embryonic days 15 and 16, there was a regional overlap in the labeling of TH and VGLUT2 mRNA in the ventral mesencephalon, which was no longer found at late embryonic stages (E18-E21) and postnatally. In normal pups from P5 to P15, only 1-2% of neurons containing TH mRNA in the ventral tegmental area (VTA) and substantia nigra, pars compacta, also displayed VGLUT2 mRNA. In contrast, after the cerebroventricular administration of 6-OHDA at P4, 26% of surviving DA neurons in the VTA of P15 rats expressed VGLUT2. To search for a colocalization of TH and VGLUT2 protein in axon terminals of these neurons, the nucleus accumbens of normal and 6-OHDA-lesioned P15 rats was examined by electron microscopy after dual immunocytochemical labeling. In normal rats, VGLUT2 protein was found in 28% of TH positive axon terminals in the core of nucleus accumbens. In 6-OHDA-lesioned rats, the total number of TH positive terminals was considerably reduced, and yet the proportion also displaying VGLUT2 immunoreactivity was modestly but significantly increased (37%). These results lead to the suggestion that the glutamatergic phenotype of a VTA DA neurons is highly plastic, repressed toward the end of normal embryonic development, and derepressed postnatally following injury. They also support the hypothesis of co-release of glutamate and DA by mesencephalic neurons in vivo, at least in the developing brain.

Bidirectional regulation of dopamine D2 and neurotensin NTS1 receptors in dopamine neurons.

Several lines of evidence suggest a close association between dopamine (DA) and neurotensin (NT) systems in the CNS. Indeed, in the rodent brain, abundant NT-containing fibres are found in DA-rich areas such as the ventral tegmental area and substantia nigra. Moreover, it has been shown in vivo that NT, acting through its high-affinity receptor (NTS1), reduces the physiological and behavioural effects of DA D2 receptor (D2R) activation, a critical autoreceptor feedback system regulating DA neurotransmission. However, the mechanism of this interaction is still elusive. The aim of our study was thus to reproduce in vitro the interaction between D2R and NTS1, and then to characterize the mechanisms implicated. We used a primary culture model of DA neurons prepared from transgenic mice expressing green fluorescent protein under the control of the tyrosine hydroxylase promoter. In these cultures, DA neurons endogenously express both D2R and NTS1. Using electrophysiological recordings, we show that activation of D2R directly inhibits the firing rate of DA neurons. In addition, we find that NT, acting through a NTS1-like receptor, is able to reduce D2R autoreceptor function independently of its ability to enhance DA neuron firing, and that this interaction occurs through a protein kinase C- and Ca(2+)-dependent mechanism. Furthermore, prior activation of D2R reduces the ability of NTS1 to induce intracellular Ca(2+) mobilization. Our findings provide evidence for bidirectional interaction between D2R and NTS1 in DA neurons, a regulatory mechanism that could play a key role in the control of the activity of these neurons.

Use of TH-EGFP transgenic mice as a source of identified dopaminergic neurons for physiological studies in postnatal cell culture.

The physiological and pharmacological properties of dopaminergic neurons in the brain are of major interest. Although much has been learned from cell culture studies, the physiological properties of these neurons remain difficult to study in such models because they are usually in minority and are difficult to distinguish from other non-dopaminergic neurons. Here we have taken advantage of a recently engineered transgenic mouse model expressing enhanced green fluorescence protein (EGFP) under the control of the tyrosine hydroxylase promoter to establish a more effective dopaminergic neuron cell culture model. We first evaluated the specificity of the EGFP expression. Although ectopic expression of EGFP was found in cultures derived from postnatal day 0 pups, this decreased over time in culture such that after 2 weeks, approximately 70% of EGFP-expressing neurons were dopaminergic. We next sought to validate this dopaminergic neuron culture model. We evaluated whether EGFP-expressing dopaminergic neurons displayed some of the well-established properties of dopaminergic neurons. Autoreceptor stimulation inhibited the activity of dopaminergic neurons while neurotensin receptor activation produced the opposite effect. Confocal imaging of the synaptic vesicle optical tracer FM4-64 in EGFP-expressing dopaminergic neurons demonstrated the feasibility of high resolution monitoring of the activity of single terminals established by these neurons. Together, this work provides evidence that primary cultures of postnatal TH-EGFP mice currently represent an excellent model to study the properties of these cells in culture.

Calcium-dependent, D2 receptor-independent induction of c-fos by haloperidol in dopamine neurons.

Antipsychotic drugs such as haloperidol act as dopamine D2 receptor antagonists to produce a number of cellular effects including the induction of immediate-early genes such as c-fos. It has been hypothesized that blockade of D2 receptors by antipsychotics is responsible for the induction of c-fos, but the mechanism has not been determined. Using cultured ventral tegmental area (VTA) dopaminergic neurons as a model, we report that nanomolar concentrations of haloperidol cause a time-dependent increase in Fos expression in dopaminergic neurons.Surprisingly, this induction was not mimicked by sulpiride, a selective D2 receptor antagonist, and was not blocked by Rp-cAMPS, an antagonist of protein kinase A (PKA), thus suggesting that D2 receptors and the cAMP cascade are not required. The induction of Fos expression was blocked by tetrodotoxin, BAPTA and KN-93, thus showing that it is activity- and calcium-dependent and requires the activation of a calmodulin-dependent kinase (CaMK). Together, these results suggest that haloperidol induces Fos expression in dopaminergic neurons through a D2 receptor-independent increase in intracellular calcium, leading to CaMK activation.

Presynaptic action of neurotensin on cultured ventral tegmental area dopaminergic neurones.

Dopamine-containing neurones of the ventral tegmental area express neurotensin receptors which are involved in regulating cell firing and dopamine release. Although indirect evidence suggests that some neurotensin receptors may be localised on the nerve terminals of dopaminergic neurones in the striatum and thus locally regulate dopamine release, a clear demonstration of such a mechanism is lacking and a number of indirect sites of action are possible. We have taken advantage of a simplified preparation in which cultured rat ventral tegmental area dopaminergic neurones establish nerve terminals that co-release glutamate to determine whether neurotensin can act at presynaptic sites. We recorded glutamate-mediated synaptic currents that were generated by dopaminergic nerve terminals as an index of presynaptic function. The neurotensin receptor agonist NT(8-13) caused an inward current and an enhancement of the firing rate of dopaminergic neurones together with an increase in the frequency of spontaneous glutamate receptor-mediated excitatory postsynaptic currents (EPSCs). Incompatible with a direct excitatory action on nerve terminals, NT(8-13) failed to change the amplitude of individual action potential-evoked EPSCs or the frequency of miniature EPSCs recorded in the presence of tetrodotoxin. However, NT(8-13) reduced the ability of terminal D2 dopamine receptors to inhibit action potential-evoked EPSCs in isolated dopaminergic neurones. Taken together, our results suggest that in addition to its well-known somatodendritic excitatory effect leading to an increase in firing rate, neurotensin also acts on nerve terminals. The main effect of neurotensin on nerve terminals is not to produce a direct excitation, but rather to decrease the effectiveness of D2 receptor-mediated presynaptic inhibition.

Comparisons of marriage and family therapists, psychologists, psychiatrists, and social workers on job-related measures and reactions to managed care in Iowa.

This study compares marriage and family therapists (MFTs) to psychologists, psychiatrists, and social workers on job-related measures, such as job autonomy, job satisfaction, burnout, and intention to stay in their present position, as well as on reactions to a managed care initiative in the state of Iowa. Findings indicate that MFTs scored significantly lower than other practitioners on job autonomy and intention to stay in their present position, but there were no differences in job satisfaction or burnout. Marital and family therapists also reported less dissatisfaction with the managed care initiative than psychiatrists, although virtually all practitioners were dissatisfied with the managed-care program. These findings indicate some dissatisfaction within the MFT profession and may be relevant to practitioners seeking to change or expand their practice, as well as to the needs of MFTs in their training programs.

Rural-urban differences in the distribution of parent-reported risk factors for substance use among young adolescents.

PURPOSE: We examined rural-urban differences in cumulative risk for youth substance use. A recent report [National Center on Addiction and Substance Abuse (CASA) 2000] found that the rural-urban distribution of substance use and known risk factors for substance use differed; in many cases rural youth showed higher levels of use, as well as higher levels of risk factors. The current investigation, while not directly examining substance use, further examined rural-urban differences in the distribution of risk factors for youth substance use, based on information from parent reports. METHOD: Study 1 data were collected from a random sample of Midwestern parents (n = 339) with a young adolescent between the ages of 11 and 13 years. Study 2 data were collected from a second sample of Midwestern parents (n = 593). RESULTS: Analyses of rural-urban comparisons demonstrated higher levels of cumulative risk among rural youth. An evaluation of the sensitivity of the analysis to rural-urban classification schemes indicated that the findings were robust, but that there was some minor variation in rural-urban differences by classification scheme. IMPLICATIONS: Results contribute to an explanation of findings from earlier reports of rural-urban differences in substance use, and suggest directions for future research on rural-urban distributions of youth risk factors.

GDNF enhances the synaptic efficacy of dopaminergic neurons in culture.

Glial cell line-derived neurotrophic factor (GDNF) is known to promote the survival and differentiation of dopaminergic neurons of the midbrain. GDNF also causes an enhancement of dopamine release by a mechanism which is presently unclear. Using isolated dopaminergic neurons of the rat ventral tegmental area in culture, we have tested the hypothesis that GDNF regulates the establishment and functional properties of synaptic terminals. Previous studies have shown that single dopaminergic neurons in culture can co-release glutamate in addition to dopamine, leading to the generation of a fast excitatory autaptic current via glutamate receptors. Using excitatory autaptic currents as an assay for the activity of synapses established by identified dopaminergic neurons, we found that chronically applied GDNF produced a threefold increase in the amplitude of excitatory autaptic currents. This action was specific for dopaminergic neurons because GDNF had no such effect on ventral tegmental area GABAergic neurons. The enhancement of excitatory autaptic current amplitude caused by GDNF was accompanied by an increase in the frequency of spontaneous miniature excitatory autaptic currents. These observations confirmed a presynaptic locus of change. We identified synaptic terminals by using synapsin-1 immunofluorescence. In single tyrosine hydroxylase-positive neurons, the number of synapsin-positive puncta which represent putative synaptic terminals was found to be approximately doubled in GDNF-treated cells at 5, 10 and 15 days in culture. The number of such morphologically identified terminals in isolated GABAergic neurons was unchanged by GDNF. These results suggest that one mechanism through which GDNF may enhance dopamine release is through promoting the establishment of new functional synaptic terminals.

Clozapine inhibits synaptic transmission at GABAergic synapses established by ventral tegmental area neurones in culture.

Elucidation of the mechanism of action of the atypical antipsychotic clozapine is complicated by the finding that this molecule interacts with multiple targets including dopaminergic and serotonergic receptors. Binding studies have suggested that clozapine also antagonises GABA(A) receptors, but physiological evidence for such a block at functional synapses is lacking. In this study, we explored this antagonism by using electrophysiological techniques on GABAergic neurones of the ventral tegmental area in culture. Inhibitory post-synaptic currents (IPSCs) evoked in isolated GABAergic neurones were found to be dose-dependently inhibited by clozapine. Compatible with a post-synaptic mechanism, we found that membrane currents evoked by exogenous applications of GABA were similarly dose-dependently inhibited by clozapine. An analysis of miniature inhibitory post-synaptic currents (mIPSCs) showed that clozapine reduced the amplitude of quantal events in a way similar to SR-95531, a specific GABA(A) receptor antagonist. Both drugs caused a similar leftward shift of the cumulative probability distribution of mIPSC amplitudes. This suggests that clozapine acts on both synaptic and extrasynaptic GABA(A) receptors. In conclusion, our work demonstrates that clozapine produces a functional antagonism of GABA(A) receptors at synapses. Because this effect occurs at concentrations that could be found in the brain of patients treated with clozapine, a reduction in GABAergic synaptic transmission could be implicated in the therapeutic actions and/or side-effects of clozapine.

Neurotensin regulates intracellular calcium in ventral tegmental area astrocytes: evidence for the involvement of multiple receptors.

Recent evidence suggests that some types of neurotensin receptors may be expressed by astrocytes. In order to explore the function of neurotensin receptors in astrocytes, the effect of a neurotensin receptor agonist, neurotensin(8-13), on intracellular Ca(2+) dynamics in mixed neuronal/glial cultures prepared from rat ventral tegmental area was examined. It was found that neurotensin(8-13) induces a long-lasting rise in intracellular Ca(2+) concentration in a subset of glial fibrilary acidic protein-positive glial cells. This response displays extensive desensitization and appears to implicate both intracellular and extracellular Ca(2+) sources. In the absence of extracellular Ca(2+), neurotensin(8-13) evokes only a short-lasting rise in intracellular Ca(2+). The neurotensin-evoked intracellular Ca(2+) accumulation is blocked by the phospholipase C inhibitor U73122 and by thapsigargin, suggesting that it is initiated by release of Ca(2+) from an inositol triphosphate-dependent store. The Ca(2+)-mobilizing action of neurotensin(8-13) in astrocytes is dependent on at least two receptors, because the response is blocked in part only by SR48692, a type 1 neurotensin receptor antagonist, and is blocked completely by SR142948A, a novel neurotensin receptor antagonist. The finding that the type 2 neurotensin receptor agonist levocabastine fails to mimic or alter the effects of neurotensin(8-13) on intracellular Ca(2+) makes it unlikely that the type 2 neurotensin receptor is involved. In summary, these results show that functional neurotensin receptors are present in cultured ventral tegmental area astrocytes and that their activation induces a highly desensitizing rise in intracellular Ca(2+). The pharmacological profile of this response suggests that a type 1 neurotensin receptor is involved but that another, possibly novel, non-type 2 neurotensin receptor is also implicated. If present in vivo, such signalling could be involved in some of the physiological actions of neurotensin.

Irbesartan: review of pharmacology and comparative properties.

The recently developed and marketed angiotensin II type 1 (AT1) receptor blockers (ARBs) have demonstrated efficacy equivalent to that of other leading classes of antihypertensive agents, with superior tolerability profiles. The specific targeting of the AT1 receptor afforded by these agents has demonstrated more complete blockade of the renin-angiotensin system than that offered by angiotensin-converting enzyme inhibitors. These data notwithstanding, differentiation within the class of ARBs has been limited. With the accumulation of additional data with ARBs, it has recently become possible to make within-class distinctions, based in large part on the individual pharmacological profiles of the ARBs. To this end, absorption, distribution, half-life, dose response and level of angiotensin II antagonism are of special note. When these properties are viewed as a group, the ARB irbesartan appears to offer advantages beyond those attained with other ARBs. Irbesartan is well absorbed, does not require biotransformation to an active metabolite to exert its antihypertensive activity, offers a large volume of distribution, has a half-life that is sufficient to allow once-daily dosing, is associated with a strong and consistent dose-response and has been demonstrated to provide a level of angiotensin II antagonism that is statistically superior to that offered by some other ARBs. These pharmacological differences may explain the clinical superiority of irbesartan compared with losartan, the first member of the ARB class. As even more data on the ARBs become available, the ability to determine the advantages of specific members of this class will be enhanced, distinctions that already have begun to come to light.

Activation of neurotransmitter release in hippocampal nerve terminals during recovery from intracellular acidification.

Intracellular pH may be an important variable regulating neurotransmitter release. A number of pathological conditions, such as anoxia and ischemia, are known to influence intracellular pH, causing acidification of brain cells and excitotoxicity. We examined the effect of acidification on quantal glutamate release. Although acidification caused only modest changes in release, recovery from acidification was associated with a very large (60-fold) increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons. This was accompanied by a block of evoked EPSCs and a rise in intracellular free Ca2+ ([Ca2+]i). The rise in mEPSC frequency required extracellular Ca2+, but influx did not occur through voltage-operated channels. Because acidic pH is known to activate the Na+/H+ antiporter, we hypothesized that a resulting Na+ load could drive Ca2+ influx through the Na+/Ca2+ exchanger during recovery from acidification. This hypothesis is supported by three observations. First, intracellular Na+ rises during acidification. Second, the elevation in [Ca2+]i and mEPSC frequency during recovery from acidification is prevented by the Na+/H+ antiporter blocker EIPA applied during the acidification step. Third, the rise in free Ca2+ and mEPSC frequency is blocked by the Na+/Ca2+ exchanger blocker dimethylbenzamil. We thus propose that during recovery from intracellular acidification a massive activation of neurotransmitter release occurs because the successive activation of the Na+/H+ and Na+/Ca2+ exchangers in nerve terminals leads to an elevation of intracellular calcium. Our results suggest that changes in intracellular pH and especially recovery from acidification have extensive consequences for the release process in nerve terminals. Excessive release of glutamate through the proposed mechanism could be implicated in excitotoxic insults after anoxic or ischemic episodes.

Modulation of an early step in the secretory machinery in hippocampal nerve terminals.

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca2+ responsiveness of the secretory machinery by partially uncoupling Ca2+-sensing from fusion per se. This is expressed as a shift toward higher Ca2+ levels of the Ca2+ to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca2+-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca2+ to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca2+ detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.

Contact-dependent regulation of N-type calcium channel subunits during synaptogenesis.

The developmental regulation of the N-type calcium channel during synaptogenesis was studied using cultured rat hippocampal neurons to elucidate the roles of extrinsic versus intrinsic cues in the expression and distribution of this channel. Prior to synapse formation, alpha1B and beta3 subunits of the N-type calcium channel were distributed diffusely throughout neurites, growth cones, and somata. As synaptogenesis proceeded, the subunit distributions became punctate and colocalized with the synaptic vesicle protein synaptotagmin. Isolated neurons were also examined to test for the requirement of extrinsic cues that control N-type calcium channel expression and distribution. These neurons expressed N-type calcium channel subunits, but their distributions remained diffuse. Functional omega-conotoxin GVIA-sensitive channels were expressed in isolated neurons, although the distribution of alpha1B subunits was diffuse. The distribution of the alpha1B subunit and synaptotagmin only became punctate when neuron-neuron contact was allowed. Thus, the expression of functional N-type calcium channels is the result of an intrinsic program while extrinsic regulatory cues mediated by neuron-neuron contact are required to control their distribution during synaptogenesis.