Cynthia Allen

Time-dependent changes in striatal glutamate synapses following a 6-hydroxydopamine lesion

The goal of this study was to investigate changes in glutamatergic synapses in the striatum of rats at two different time-points following a unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. One month following this lesion of the nigrostriatal pathway, there was an increase (70%) in the mean percentage of asymmetrical synapses within the dorsolateral striatum containing a discontinuous, or perforated, postsynaptic density, possibly suggesting an increase in glutamatergic activity. This was correlated, in the same brain region, with a decrease (44%) in the density of glutamate immunoreactivity within nerve terminals associated with all asymmetrical synapses and also with those terminals associated with a perforated postsynaptic density. These morphological changes were consistent with an increase (>two-fold) in the basal extracellular level of striatal glutamate, as measured by in vivo microdialysis. The density of GABA immunolabeling within symmetrical nerve terminals was increased (25%) at this one month time-period. Dopamine levels within the lesioned striatum were >99% depleted. However, at three months, while an increase in the mean percentage of striatal perforated synapses was maintained, a significant increase (50%) in the density of striatal nerve terminal glutamate immunolabeling within all asymmetrical synapses and those associated with a perforated postsynaptic density was observed. This was correlated with a small, but significant, decrease (32%) in the basal extracellular level of striatal glutamate. The density of GABA immunolabeling within nerve terminals associated with a symmetrical contact remained elevated at this three month time-period, while striatal dopamine levels remained depleted. While the density of nerve terminal GABA immunolabeling remained elevated at both the one and three month time-periods, there appeared to be a differential effect on glutamatergic synapses. The in vivo microdialysis data suggest that glutamate synapses were more active at a basal level at one month and become less active compared to the control group at the three month time-period. These data suggest that there are compensatory changes in glutamatergic synapses within the striatum following a 6-hydroxydopamine lesion that appear to be independent of the level of striatal dopamine or GABA. We propose that changes in the activity of the thalamo-cortico-striatal pathway may help to explain the differential time-course change in striatal glutamatergic synaptic activity.

PRESYNAPTIC INHIBITION BY BACLOFEN OF RETINOHYPOTHALAMIC EXCITATORY SYNAPTIC TRANSMISSION IN RAT SUPRACHIASMATIC NUCLEUS

Optic nerve stimulation evoked monosynaptic excitatory postsynaptic currents in suprachiasmatic nucleus neurons maintained in vitro. These currents were completely blocked by a combination of glutamate receptor antagonists, 6-cyano-7-nitroquinoxaline-2,3-dione and 4-aminophosphonovaleric acid. Stimulation of the ipsilateral or contralateral suprachiasmatic nucleus produced a biphasic response consisting of an excitatory postsynaptic current followed by an bicuculline-sensitive inhibitory postsynaptic current. Most suprachiasmatic nucleus neurons had spontaneous inhibitory and excitatory synaptic currents produced by action potential-independent and, less frequently, action potential-dependent release of GABA and glutamate. Baclofen reversibly reduced the amplitude of excitatory postsynaptic currents evoked by optic nerve stimulation and the effect was antagonized by 2-hydroxysaclofen. In addition, baclofen reduced the frequency but not the amplitude of the spontaneous miniature excitatory postsynaptic currents. In a subset of suprachiasmatic nucleus neurons, baclofen induced an outward current, probably by increasing a potassium conductance. Baclofen had no effect on either evoked or spontaneous inhibitory postsynaptic currents or on currents activated by pulse application of glutamate. These data indicate that activation of GABAB receptors can inhibit suprachiasmatic nucleus neurons by two mechanisms. The first is to inhibit the release of glutamate from terminals of the retinohypothalamic tract. The second is the postsynaptic activation of a potassium conductance in a portion of these neurons.

Effects of subchronic clozapine and haloperidol on striatal glutamatergic synapses

Abstract: Subchronic treatment with haloperidol increases the number of asymmetric glutamate synapses associated with a perforated postsynaptic density in the striatum. To characterize these synaptic changes further, the effects of subchronic (28 days) administration of an atypical antipsychotic, clozapine (30 mg/kg, s.c.), or a typical antipsychotic, haloperidol (0.5 mg/kg, s.c.), on the binding of [3H]MK‐801 to the NMDA receptor‐linked ion channel complex and on the in situ hybridization of riboprobes for NMDAR2A and 2B subunits and splice variants of the NMDAR1 subunit were examined in striatal preparations from rats. The density of striatal glutamate immunogold labeling associated with nerve terminals of all asymmetric synapses and the immunoreactivity of those asymmetric synapses associated with a perforated postsynaptic density were also examined by electron microscopy. Subchronic neuroleptic administration had no effect on [3H]MK‐801 binding to striatal membrane preparations. Both drugs increased glutamate immunogold labeling in nerve terminals of all asymmetric synapses, but only haloperidol increased the density of glutamate immunoreactivity within nerve terminals of asymmetric synapses containing a perforated postsynaptic density. Whereas subchronic administration of clozapine, but not haloperidol, resulted in a significant increase in the hybridization of a riboprobe that labels all splice variants of the NMDAR1 subunit, both drugs significantly decreased the abundance of NMDAR1 subunit mRNA containing a 63‐base insert. Neither drug altered mRNA for the 2A subunit, but clozapine significantly increased hybridization of a probe for the 2B subunit. The data suggest that some neuroleptic effects may be mediated by glutamatergic systems and that typical and atypical antipsychotics can have varying effects on the density of glutamate in presynaptic terminals and on the expression of specific NMDA receptor splice variant mRNAs. Alternatively, NMDAR1 subunit splice variants may differentially respond to interactions with glutamate.

Potential pathways for intercellular communication within the calbindin subnucleus of the hamster suprachiasmatic nucleus.

In mammals, the suprachiasmatic nucleus (SCN) is the master circadian pacemaker. Within the caudal hamster SCN, a cluster of neurons containing the calcium binding protein, calbindin-D28K (CB), has been implicated in circadian locomotion. However, calbindin-immunoreactive (CB+) neurons in the calbindin subnucleus (CBsn) do not display a circadian rhythm in spontaneous firing [Eur J Neurosci 16 (2002) 2469]. Previously, we proposed that intercellular communication might be essential in integrating outputs from rhythmic (CB-) neurons and nonrhythmic (CB+) neurons to produce a circadian output in the intact animal. The primary aim of this study is to provide a neuroanatomical framework to better understand intercellular communication within the CBsn. Using reconstructions of previously recorded neurons, we demonstrate that CB+ neurons have significantly more dendrites than CB- neurons. In addition, CBsn neurons have dorsally oriented dendritic arbors. Using double-label confocal microscopy, we show that GABA colocalizes with CB+ neurons and GABA(A) receptor subunits make intimate contacts with neurons in the CBsn. Transforming growth factor alpha (TGFalpha), a substance shown to inhibit locomotion [Science 294 (2001) 2511], is present within the CBsn. In addition, neurons in this region express the epidermal growth factor receptor, the only receptor for TGFalpha. Lastly, we show that CB+ neurons are coupled to CB+ and CB- neurons by gap junctions. The current study provides a structural framework for synaptic communication, electrical coupling, and signaling via a growth factor within the CBsn of the hamster SCN. Our results reveal connections that have the potential for integrating cellular communication within a subregion of the SCN that is critically involved in circadian locomotion.

Characterization of an apamin-sensitive potassium current in suprachiasmatic nucleus neurons

In neurons of the suprachiasmatic nucleus, spike frequency adaptation and membrane afterhyperpolarization occur during a train of action potentials. Extracellular Ca2+ may regulate neuronal excitability by several mechanisms, including activation of small conductance and large conductance Ca(2+)-activated K+ channels. The overall goal of this study was to examine the role of Ca(2+)-activated K+ currents in individual suprachiasmatic nucleus neurons. To this end, we used the nystatin-perforated patch technique to record currents from suprachiasmatic nucleus neurons. Iberiotoxin and tetraethylammonium, antagonists of large conductance Ca(2+)-activated K+ channels, had no effect on the membrane afterhyperpolarization. However, antagonists of small conductance Ca(2+)-activated K+ channels, apamin and d-tubocurarine, reduced the amplitude of the membrane afterhyperpolarization and inhibited the spike frequency adaptation that occurred during a train of action potentials. Although there was no significant difference in membrane AHP between different portions of the circadian day, apamin and d-tubocurarine increased the spontaneous firing frequency of suprachiasmatic nucleus neurons during the daytime. In voltage-clamp mode, membrane depolarization-activated currents were followed by an outward tail current reversing near the K+ equilibrium potential. The tail current decayed with a time constant of 220 ms at +20 mV and 149 ms at -40 mV. Apamin irreversibly and d-tubocurarine reversibly inhibited the tail current. The tail current amplitude was also reduced by the GABAA receptor antagonist, bicuculline methiodide, while picrotoxin (another GABAA receptor antagonist) was without effect. Removal of extracellular Ca2+ or the addition of Cd2+ reversibly inhibited the tail current. These results indicate that apamin- and d-tubocurarine-sensitive small conductance Ca(2+)-activated K+ channels have a modulatory function on the action potential firing frequency as well as the membrane afterhyperpolarization that follows a train of action potentials in suprachiasmatic nucleus neurons. Importantly, our data also indicate that a portion of the effects of bicuculline methiodide on suprachiasmatic nucleus neurons may be mediated by inhibition of small conductance Ca(2+)-activated K+ channels.

Haloperidol reverses the changes in striatal glutamatergic immunolabeling following a 6-OHDA lesion.

We reported previously that 3 months following a unilateral lesion of the nigrostriatal pathway with 6‐hydroxydopamine (6‐OHDA), there was a decrease in the extracellular level of striatal glutamate as determined by in vivo microdialysis. This resulted in an accumulation or increase in the density of nerve terminal glutamate immunolabeling (Meshul et al., 1999). We also reported on blockade of dopamine D‐2 receptors with haloperidol resulting in ultrastructural changes within the striatum consistent with increased functioning of the glutamatergic corticostriatal pathway (Meshul and Tan 1994). We hypothesized that administration of haloperidol to 6‐OHDA‐lesioned rats may be capable of activating the corticostriatal pathway and thereby counteracting the effects of the unilateral nigrostriatal lesion. Striatal glutamatergic function was evaluated using electron microscopy and quantitative glutamate immunocytochemistry. Starting 1 month after a unilateral lesion of the nigrostriatal pathway with 6‐OHDA, haloperidol (0.5 mg/kg/d) was administered for the next 2 months. Within the dorsolateral caudate nucleus, the main area of innervation from the motor cortex, haloperidol blocked the 6‐OHDA‐induced increase in the density of nerve terminal glutamate immunolabeling. Within all three experimental groups (6‐OHDA, haloperidol, 6‐OHDA/haloperidol) there was an increase in the mean percentage of striatal asymmetrical synapses containing a perforated postsynaptic density. In addition, haloperidol treatment resulted in a reduction in the number of apomorphine‐induced contralateral rotations in unilaterally 6‐OHDA lesioned rats. The data suggests that the decrease in striatal glutamatergic function 3 months following a unilateral 6‐OHDA lesion can be reversed by daily haloperidol treatment. This finding is discussed in terms of current therapy for Parkinsons disease. Synapse 36:129–142, 2000. Published 2000 Wiley‐Liss, Inc.

Activation of corticostriatal pathway leads to similar morphological changes observed following haloperidol treatment.

Treatment with haloperidol, a dopamine receptor D‐2 antagonist, for one month resulted in an increase in the mean percentage of asymmetric synapses containing a discontinuous, or perforated, postsynaptic density (PSD) [Meshul et al. (1994) Brain Res., 648:181–195] and a change in the density of striatal glutamate immunoreactivity within those presynaptic terminals [Meshul and Tan (1994) Synapse, 18:205–217]. We speculated that this haloperidol‐induced change in glutamate density might be due to an activation of the corticostriatal pathway. To determine if activation of this pathway leads to similar morphological changes previously described following haloperidol treatment, GABA (10−5 M, 0.5 μl) was injected into the thalamic motor (VL/VM) nuclei daily for 3 weeks. This treatment resulted in an increase in the mean percentage of striatal asymmetric synapses containing a perforated PSD and an increase in the density of glutamate immunoreactivity within nerve terminals of asymmetric synapses containing a perforated or non‐perforated PSD. Subchronic injections of GABA into the thalamic somatosensory nuclei (VPM/VPL) had no effect on the mean percentage of synapses with perforated PSDs but resulted in a small, but significant, increase in density of glutamate immunoreactivity. Using in vivo microdialysis, an acute injection of GABA (10−5 M, 15 μl) into VL/VM resulted in a prolonged rise in the extracellular level of striatal glutamate. The increase in asymmetric synapses with perforated PSDs and in glutamate immunoreactivity within nerve terminals of the striatum following either subchronic haloperidol treatment or GABA injections into VL/VM suggest that an increase in glutamate release may be a common factor in these two experiments. It is possible that the extrapyramidal side effects associated with haloperidol treatment may be due, in part, to an increase in release of glutamate within the corticostriatal pathway.

Nociceptin/orphanin FQ (N/OFQ) inhibits excitatory and inhibitory synaptic signaling in the suprachiasmatic nucleus (SCN)

Environmental synchronization of the endogenous mammalian circadian rhythm involves glutamatergic and GABAergic neurotransmission within the hypothalamic suprachiasmatic nucleus (SCN). The neuropeptide nociceptin/orphanin FQ (N/OFQ) inhibits light-induced phase shifts, evokes K(+)-currents and reduces the intracellular Ca(2+) concentration in SCN neurons. Since these effects are consistent with a modulatory role for N/OFQ on synaptic transmission in the SCN, we examined the effects of N/OFQ on evoked and spontaneous excitatory and inhibitory currents in the SCN. N/OFQ produced a consistent concentration-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSC) evoked by optic nerve stimulation. N/OFQ did not alter the amplitude of currents induced by application of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-d-aspartate (NMDA) nor the amplitude of miniature EPSC (mEPSC) consistent with a lack of N/OFQ effect on postsynaptic AMPA or NMDA receptors. N/OFQ significantly reduced the mEPSC frequency. The inhibitory actions of N/OFQ were blocked by omega-conotoxin GVIA, an N-type Ca(2+)channel antagonist and partially blocked by omega-agatoxin TK, a P/Q type Ca(2+) channel blocker. These data indicate that N/OFQ reduces evoked EPSC, in part, by inhibiting the activity of N- and P/Q-type Ca(2+) channels. In addition, N/OFQ produced a consistent reduction in baseline Ca(2+) levels in presynaptic retinohypothalamic tract terminals. N/OFQ also inhibited evoked GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSC) in a concentration dependent manner. However, N/OFQ had no effect on currents activated by muscimol application or on the amplitude of miniature IPSC (mIPSC) and significantly reduced the mIPSC frequency consistent with an inhibition of GABA release downstream from Ca(2+) entry. Finally, N/OFQ inhibited the paired-pulse depression observed in SCN GABAergic synapses consistent with a presynaptic mechanism of action. Together these results suggest a widespread modulatory role for N/OFQ on the synaptic transmission in the SCN.

β-N-Methylamino-l-alanine in the presence of bicarbonate is an agonist at non- N-methyl-d-aspartate-type receptors

Beta-N-Methylamino-L-alanine, a component of the neurotoxic Cycas circinalis plant, activates an ionic current which is antagonized by extracellular Ca2+ but not by the excitatory amino acid receptor antagonists D,L-2-amino-5-phosphonovalerate (10-100 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione (1-10 microM). This current was reduced by 50% in 0.5 mM extracellular Ca2+ and 92% in 3.0 mM Ca2+ when compared to those recorded in 0.1 mM Ca2+. Addition of 10 or 20 mM NaHCO3 to beta-N-methylamino-L-alanine (500 microM) potentiated the currents 224% and 578%, respectively. Addition of NaHCO3 to the extracellular Ringers (pH 7.2) shifted the pH to 7.7 (10 mM) or 8.3 (20 mM). beta-N-Methylamino-L-alanine was potentiated by NaHCO3 at pH 7.2, 7.7 and 8.3, but the potentiation with NaHCO3 (20 mM) was larger at pH 8.3 (5.7-fold) compared to pH 7.2 (3-fold). NaHCO3 (20 mM) had no effect on quisqualate-, N-methyl-D-aspartate- or kainate-activated ionic currents. The beta-N-methylamino-L-alanine-NaHCO3-activated currents were reduced 49% by 1 microM and 80% by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione suggesting an agonist action at non-N-methyl-D-aspartate-type receptors. Activity at N-methyl-D-aspartate receptors is unlikely since the beta-N-methylamino-L-alanine-NaHCO3 currents are not antagonized by D,L-2-amino-5-phosphonovalerate (10-100 microM), potentiated by addition of glycine (10 microM) or blocked by extracellular Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

The sulphydryl reagent, N-ethylmaleimide, disrupts sleep and blocks A1 adenosine receptor-mediated inhibition of intracellular calcium signaling in the in vitro ventromedial preoptic nucleus

To explore the neuronal signaling mechanisms underlying sleep regulation in the rat, the present study examined continuous intra-third ventricle infusion of N-ethylmaleimide (NEM), a sulphydryl reagent that inhibits G(i/o) protein-coupled receptor-mediated signaling pathways. The diurnal infusion of NEM (0.01-10 micromol/10 h) dose-dependently inhibited both non-rapid eye movement sleep and rapid eye movement sleep. A maximal dose of NEM (10 micromol/10 h) dramatically inhibited day-time sleep (-57% for non-rapid eye movement sleep and -89% for rapid eye movement sleep) with a compensatory increase of sleep during the subsequent night-time (+33% for non-rapid eye movement sleep and +259% for rapid eye movement sleep). The day-time brain temperature was also increased by NEM, demonstrating effects of NEM on both sleep and body temperature levels. Immunostaining of the rat hypothalamus with a monoclonal antibody against the A1 adenosine receptor (A1R) was used to explore the distribution of a sleep-related G(i/o) protein-coupled receptor. Robust A1R-like immunoreactivity was found in the ventromedial preoptic nucleus and the supraoptic nucleus. Fura-2-based Ca(2+) imaging analysis of acute hypothalamic slices further demonstrated that the A1R agonist N(6)-cyclopentyladenosine (CPA; 200 nM) inhibited spontaneous Ca(2+) oscillations and high potassium (80 mM)-induced Ca(2+) flux in the ventromedial preoptic nucleus, while NEM (100-300 microM) and an A1R antagonist 8-cyclopentyl-dipropylxanthine (300 nM) blocked the CPA actions and increased the high potassium-induced Ca(2+) flux. From these results we suggest that NEM-sensitive G protein-coupled receptor(s) may play an important role in the regulation of sleep and body temperature in the rat and one possible mechanism is an A1R-mediated regulation of intracellular Ca(2+) concentrations in the ventromedial preoptic nucleus.