Noradrenaline mediates slow excitatory synaptic potentials in rat dorsal raphe neurons in vitro

Repetitive focal stimulation to the slice surface within the region of the dorsal raphe (DR) nucleus of rat brain elicited a slow excitatory synaptic potential (slow EPSP), which followed a slow inhibitory synaptic potential (slow IPSP) in a majority of the DR neurons. The slow EPSPs were associated with either an increase of a decrease in membrane resistance. Noradrenaline (NA) application caused a membrane depolarization in most of the DR neurons. The NA-induced depolarization was also accompanied by either an increase or a decrease in membrane resistance. Both the slow EPSP and NA-induced depolarization were inhibited by phentolamine and prazosin but not by yohimbine and propranolol. The result suggests that slow EPSPs in rat DR neurons are mediated by NA interacting with an alpha 1-adrenoceptor.     

GABA- and glutamate-mediated synaptic potentials in rat dorsal raphe neurons in vitro

1. Synaptic potentials were recorded with intracellular electrodes from rat dorsal raphe neurons in a slice preparation. 2. Synaptic potentials were evoked by applying electrical pulses to bipolar stimulating electrodes positioned immediately dorsal to the raphe nucleus; these arose after a latency of 0.5-5 ms and had a duration of 20-200 ms. 3. The synaptic potential was biphasic (at the resting potential) when the recording electrodes contained potassium citrate; a depolarization was followed by a hyperpolarization. The hyperpolarization reversed in polarity at -70 mV and was blocked by bicuculline. 4. The depolarizing synaptic potential was reduced to 50-90% of control by kynurenate (1-2 mM) or 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX) (10 microM) and increased in amplitude and duration by magnesium-free solution. 5. In magnesium-free solutions (with CNQX), the depolarizing synaptic potential was blocked by DL-2-amino-5-phosphonovaleric acid (APV, 50 microM). APV also blocked depolarization caused by adding N-methyl-D-aspartate (NMDA) to the superfusion solution. 6. The results indicate that raphe neurons display two synaptic potentials having a duration of 150-200 ms: one that is mediated by GABA and a second that is due to an excitatory amino acid. The component mediated by an excitatory amino acid involves, in part, a receptor of the NMDA type.     

Fractalkine/CX3CL1 enhances GABA synaptic activity at serotonin neurons in the rat dorsal raphe nucleus

Serotonin (5-hydroxytryptamine; 5-HT) has an important role in mood regulation, and its dysfunction in the central nervous system (CNS) is associated with depression. Reports of mood and immune disorder co-morbidities indicate that immune-5-HT interactions may mediate depression present in immune compromised disease states including HIV/AIDS, multiple sclerosis, and Parkinson's disease. Chemokines, immune proteins that induce chemotaxis and cellular adhesion, and their G-protein coupled receptors distribute throughout the CNS, regulate neuronal patterning, and mediate neuropathology. The purpose of this study is to investigate the neuroanatomical and neurophysiological relationship between the chemokine fractalkine/CX3CL1 and its receptor CX3CR1 with 5-HT neurons in the rat midbrain raphe nuclei (RN). Immunohistochemistry was used to examine the colocalization of CX3CL1 or CX3CR1 with 5-HT in the RN, and whole-cell patch-clamp recordings in rat brain slices were used to determine the functional impact of CX3CL1 on 5-HT dorsal raphe nucleus (DRN) neurons. Greater than 70% of 5-HT neurons colocalize with CX3CL1 and CX3CR1 in the RN. CX3CL1 localizes as discrete puncta throughout the cytoplasm, whereas CX3CR1 concentrates to the perinuclear region of 5-HT neurons and exhibits microglial expression. CX3CL1 and CX3CR1 also colocalize with one another on individual RN cells. Electrophysiology studies indicate a CX3CL1-mediated enhancement of spontaneous inhibitory postsynaptic current (sIPSC) amplitude and dose-dependent increase of evoked IPSC (eIPSC) amplitude without affecting eIPSC paired–pulse ratio, a finding observed selectively in 5-HT neurons. CX3CL1's effect on eIPSC amplitude is blocked by pretreatment with an anti-CX3CL1 neutralizing antibody. Thus, CX3CL1 enhances postsynaptic GABA receptor number or sensitivity on 5-HT DRN neurons under conditions of both spontaneous and synaptically-evoked GABA release. CX3CL1 may indirectly inhibit 5-HT neurotransmission by increasing the sensitivity of 5-HT DRN neurons to GABA inputs. Therapies targeting CX3CL1 may treat serotonin related mood disorders, including depression experienced by patients with compromised immune systems.     

Participation of excitatory amino acid receptors in the slow excitatory synaptic transmission in the rat spinal dorsal horn in vitro

The participation of excitatory amino acid (EAA) receptors in the responses of deep dorsal horn neurons to repetitive stimulation of dorsal roots was investigated using a spinal slice preparation and current-clamp and voltage-clamp techniques. Using EAA receptor and substance P (SP) receptor antagonists and current-clamp, slow excitatory synaptic response evoked by 10-20 Hz stimulation consisted of two depolarizing components: an initial component lasting 1-5 s and a late-one of 1-3 min duration. The initial and late components of the slow excitatory postsynaptic currents (EPSCs) can also be distinguished on the basis of their voltage-dependence and sensitivity to Mg2+ ions, D-2-amino-5-phosphonovalerate (D-APV) and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX). In the presence of Mg2+, the initial component of the slow EPSC increased with membrane hyperpolarization, whereas the late component decreased. In a zero-Mg2+ medium, the initial component was potentiated, but the late component was reduced, or unchanged. CNQX reduced the initial component. In a zero-Mg2+ solution, or at membrane potentials positive to -55 mV in 1 mM Mg2+, D-APV reduced or even abolished the initial component, whereas the late component was not modified by D-APV. We propose that slow excitatory synaptic response evoked in deep dorsal horn neurons by repetitive stimulation of primary afferents has two components, an initial transient component that requires activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, and a late longer-lasting peptidergic component that has been already described (Brain Res., 290 (1984) 336-341.     

Age-related alteration in excitatory amino acid neurotransmission in rat brain

The excitatory amino acids as neurotransmitters in the neocortex, hippocampus, striatum, thalamus, amygdala, nucleus basalis of Meynert and cerebellum from rats aged 4 months, 12 months and 24 months have been examined by measuring sodium-dependent high affinity uptake of D-[3H]-aspartate into preparations containing synaptosomes. Calcium-dependent K(+)-stimulated release of endogenous glutamate from the nucleus basalis was also measured. The hippocampus and cerebellum failed to show significant age-related changes in uptake of D-[3H]-aspartate. D-[3H]-aspartate uptake decreased significantly in the neocortex (29%), striatum (29%), nucleus basalis (26%), amygdala (19%) and thalamus (16%) in the middle-aged rats as compared to the young rats, but the changes were not progressive with age. The release of glutamate from the nucleus basalis was unaltered during the aging process.     

The lateral habenula regulates defensive behaviors through changes in 5-HT-mediated neurotransmission in the dorsal periaqueductal gray matter

Chemical stimulation of the lateral nucleus of the habenula (LHb), an area implicated in the regulation of serotonergic activity in raphe nuclei, affects the acquisition of inhibitory avoidance and escape expression of rats submitted to the elevated T-maze test of anxiety. Here, we investigated whether facilitation of 5-HT-mediated neurotransmission in the dorsal periaqueductal gray (dPAG) accounts for the behavioral consequences in the elevated T-maze induced by chemical stimulation of the LHb. The dPAG in the midbrain, which is innervated by 5-HT fibers originating from the dorsal raphe nucleus (DRN), has been consistently implicated in the genesis/regulation of anxiety- and fear-related defensive responses. The results showed that intra-dPAG injection of WAY-100635 or ketanserin, 5-HT_1A and 5-HT_2A/2C receptor antagonists, respectively, counteracted the anti-escape effect caused by bilateral intra-LHb injection of kainic acid (60 pmol/0.2 μl). Ketanserin, but not WAY-100635, blocked kainic acid's facilitatory effect on inhibitory avoidance acquisition. Overall, the results suggest that the pathway connecting the LHb to the DRN is involved in the control of 5-HT release in the dPAG, and facilitation of 5-HT-mediated neurotransmission in the latter area distinctively impacts upon the expression of anxiety- and fear-related defensive behaviors. While stimulation of 5-HT_1A receptors selectively affects escape performance, 5-HT_2A/2C receptors modulate both inhibitory avoidance and escape.     

Opioidergic, GABAergic and serotonergic neurotransmission in the dorsal raphe nucleus modulates tonic immobility in guinea pigs

Tonic immobility (TI) is an innate defensive behavior that can be elicited by physical restriction and postural inversion and is characterized by a profound and temporary state of akinesis. Our previous studies demonstrated that the stimulation of serotonin receptors in the dorsal raphe nucleus (DRN) appears to be biphasic during TI responses in guinea pigs (Cavia porcellus). Serotonin released by the DRN modulates behavioral responses and its release can occur through the action of different neurotransmitter systems, including the opioidergic and GABAergic systems. This study examines the role of opioidergic, GABAergic and serotonergic signaling in the DRN in TI defensive behavioral responses in guinea pigs. Microinjection of morphine (1.1 nmol) or bicuculline (0.5 nmol) into the DRN increased the duration of TI. The effect of morphine (1.1 nmol) was antagonized by pretreatment with naloxone (0.7 nmol), suggesting that the activation of μ opioid receptors in the DRN facilitates the TI response. By contrast, microinjection of muscimol (0.5 nmol) into the DRN decreased the duration of TI. However, a dose of muscimol (0.26 nmol) that alone did not affect TI, was sufficient to inhibit the effect of morphine (1.1 nmol) on TI, indicating that GABAergic and enkephalinergic neurons interact in the DRN. Microinjection of alpha-methyl-5-HT (1.6 nmol), a 5-HT(2) agonist, into the DRN also increased TI. This effect was inhibited by the prior administration of naloxone (0.7 nmol). Microinjection of 8-OH-DPAT (1.3 nmol) also blocked the increase of TI promoted by morphine (1.1 nmol). Our results indicate that the opioidergic, GABAergic and serotonergic systems in the DRN are important for modulation of defensive behavioral responses of TI. Therefore, we suggest that opioid inhibition of GABAergic neurons results in disinhibition of serotonergic neurons and this is the mechanism by which opioids could enhance TI. Conversely, a decrease in TI could occur through the activation of GABAergic interneurons.     

Presynaptic NMDA receptor-mediated modulation of excitatory neurotransmission in the mouse dorsal motor nucleus of the vagus

Activity of neurons in the dorsal motor nucleus of the vagus nerve (DMV) is closely regulated by synaptic input, and regulation of that input by glutamate receptors on presynaptic terminals has been proposed. Presynaptic N-methyl-d-aspartic acid (NMDA) receptors have been identified in a number of brain regions and act to modulate neurotransmitter release, but functional presynaptic NMDA receptors have not been adequately studied in the DMV. This study identified the presence and physiological function of presynaptic NMDA receptors on synaptic input to DMV neurons. Whole-cell patch-clamp recordings from DMV neurons in acute slices from mice revealed prevalent miniature excitatory postsynaptic currents, which were significantly increased in frequency, but not amplitude, by application of NMDA. Antagonism of NMDA receptors with dl-2-amino-5-phosphonopentanoic acid (100 μM) resulted in a decrease in miniature excitatory postsynaptic current frequency and an increase in the paired pulse ratio of responses following afferent stimulation. No consistent effects of presynaptic NMDA receptor modulation were observed on GABAergic inputs. These results suggest that presynaptic NMDA receptors are present in the dorsal vagal complex and function to facilitate the release of glutamate, preferentially onto DMV neurons tonically, with little effect on GABA release. This type of presynaptic modulation represents a potentially novel form of glutamate regulation in the DMV, which may function to regulate glutamate-induced activity of central parasympathetic circuits.     

Calcium dependent aspects of synaptic plasticity, excitatory amino acid neurotransmission, brain aging and schizophrenia: a unifying hypothesis

(1) The functional and structural reorganization of dendritic spines by calcium activated proteases is postulated to play a causal role in the production of the phenomenology of brain aging and in particular in the development of pathology and degeneration. Excitatory neurotransmission appears to be essential for the development of irreversible synaptic changes. (2) One of the genes modified in schizophrenia is postulated to be directly or indirectly linked to the control of excitatory neurotransmission; possibly the normal switching on of the expression of the adult form of the NMDA receptor is altered, resulting in an inappropriate functioning of this receptor. This genetic characteristic might explain the apparent resistance of schizophrenic brains to aging.     

Autoradiographic and electrophysiological evidence for excitatory amino acid transmission in the periaqueductal gray projection to nucleus raphe magnus in the rat

Selective retrograde labelling was used as an autoradiographic method to identify possible excitatory amino acid afferents to nucleus raphe magnus (NRM). Injections of 25-50 nl 10(-2) or 10(-3) M D-[3H]aspartate into the NRM resulted in prominent labelling of cells in ventrolateral mesencephalic periaqueductal gray (PAG). Electrophysiologically, stimulation in ventrolateral PAG excited cells in NRM with a latency of 2-12 ms. With many cells, microelectrophoretic application of the excitatory amino acid antagonists, kynurenate and gamma-D-glutamyl-glycine, resulted in a reversible reduction of the PAG-evoked response. Selective antagonists of N-methyl-D-aspartate (NMDA) were less effective. It is suggested that neurones in the ventrolateral PAG projecting to NRM utilize an excitatory amino acid or structurally related compound as a transmitter, and that this transmitter acts on receptors of the non-NMDA type.     

Kainic acid microinjected into the cat raphe dorsal nucleus modulates the somatosensory evoked potentials and their cycles of excitability

Studies were made on the effect of the neuroexcitatory agent kainic acid, microinjected into raphe dorsal nucleus by glass micropipette and an air pressure system in doses ranging from 0.2 to 24.0 nmol (in volumes from 0.05 microliter to 0.47 microliter), on the somatosensory evoked potentials and their cycles of recovery (excitability) obtained from cortex (primary somatosensory and parietal associative), thalamus (ventral posterolateral nucleus and centre median nucleus), mesencephalic reticular formation and raphe dorsal nucleus. Kainic acid in doses higher than 3 nmol exerted an activating effect on the evoked potentials and their recovery cycles especially in thalamus and mesencephalic reticular formation. The analysis of these electrophysiological parameters revealed that the non-specific structures were involved to a larger extent in the activating effect of kainic acid than the specific ones. The morphological changes were not severe and were limited to a part of the raphe dorsal nucleus neurons. Our data indicate that kainic acid injected into raphe dorsal nucleus modulates (in direction of facilitation) the somatosensory evoked potentials and their cycles of excitability obtained in some brain structures. The results suggest that this nucleus is involved in the somatosensory information processing in a non-specific manner.     

Endogenous GABA mediates presynaptic inhibition of spontaneous and evoked excitatory synaptic potentials in the rat neostriatum

The effect of the blockade of the gamma-aminobutyric acid (GABA) uptake system on the amplitude of glutamatergic synaptic potentials was studied by using a corticostriatal slice preparation. Nipecotic acid (0.1-1 mM), a GABA uptake blocker, produced a dose-dependent decrease of the amplitude of kynurenate-sensitive excitatory synaptic potentials recorded in the neostriatum following cortical stimulation. Nipecotic acid did not affect the postsynaptic responses to exogenously applied glutamate. The presynaptic effect of endogenous GABA was bicuculline-resistant and was mimicked by baclofen (0.3-3 microM). This effect was not blocked by phaclofen (0.5-1 mM). These findings show that phaclofen-insensitive GABAB receptors, activated by endogenous GABA, mediate presynaptic inhibition of cortical glutamatergic inputs in the neostriatum.     

Effect on memory processes of anti-vasopressin serum microinjected into the dorsal raphe nucleus: The role of catecholaminergic neurotransmission

Summary Anti-arginine⁸-vasopressin serum was microinjected into the mesencephalic dorsal raphe nucleus immediately after the learning trial, in a one-trial learning passive avoidance reaction. The treatment attenuated passive avoidance behavior 24 h after treatment, suggesting a role of the endogenous vasopressin of this area in memory processes. On the other hand, the antiserum did not influence passive avoidance behavior if 6-hydroxydopamine was microinjected into the raphe region. The data suggest that the antiserum may have primarily interacted with catecholaminergic terminals, which enter the dorsal raphe nucleus.This work was supported by the Scientific Research Council, Ministry of Health, Hungary [Grant No. 4-08-0302-03-0(T)]     

K+ channel and 5-hydroxytryptamine1A autoreceptor interactions in the rat dorsal raphe nucleus: an in vitro electrophysiological study.

Extracellular recordings were made from serotonergic neurons of the rat dorsal raphe nucleus in a slice preparation. In the presence of phenylephrine (3 microM) to restore the pacemaker activity of otherwise silent serotonergic neurons, superfusion with the 5-hydroxytryptamine1A agonist ipsapirone depressed the firing of these neurons with an IC50 of approximately 50 nM. Complete inhibition was achieved with 100-300 nM of the drug. Concomitant superfusion with the 5-hydroxytryptamine1A antagonists spiperone (100 nM) or propranolol (10 microM) markedly reduced the inhibitory effect of ipsapirone (100 nM). Superfusion with K+ channel blockers such as apamin (50-100 nM), charybdotoxin (100 nM) or Ba2+ (1 mM) did not induce any changes in the electrical activity of serotonergic neurons. However, 4-aminopyridine (0.1-1 mM) disrupted the regularity of their discharge without affecting the mean firing rate. The ipsapirone-induced inhibition was unchanged by apamin and charybdotoxin, but was markedly reduced by Ba2+ and 4-aminopyridine. Thus the IC50 of ipsapirone was shifted to approximately 150 nM in the presence of 1 mM of 4-aminopyridine. These results indicate that, in serotonergic neurons within the dorsal raphe nucleus, the K+ channel opened through the stimulation of 5-hydroxytryptamine1A autoreceptors is 4-aminopyridine-sensitive.     

Actions of excitatory amino acid antagonists on synaptic inputs to the rat medial vestibular nucleus: an electrophysiological study in vitro

Summary The actions of excitatory amino acid (EAA) antagonists on synaptic inputs to neurons in the rat medial vestibular nucleus (MVN) from ipsilateral vestibular afferents and vestibular commissures were studied in brain stem slice preparations. Antagonists used were 2-amino-5-phosphonovalerate (APV), a selective antagonist for the N-methyl-D-aspartate (NMDA) type of EAA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective antagonist for the quisqualate/kainate (non-NMDA) type of EAA receptors and kynurenate (KYNA), a broad spectrum antagonist for the three types of EAA receptors. MVN neurons were classified as having mono- or polysynaptic inputs from vestibular afferents and commissural fibers by calculating synaptic delay. An application of APV through the perfusion medium suppressed 82% of cells activated monosynaptically from commissures, while it suppressed only 9% of cells activated monosynaptically from vestibular afferents. The application of KYNA proved much less selective, suppressing 83% of the former group of cells and 93% of the latter. CNQX suppressed almost all the cells of both groups. The sensitivity of monosynaptic inputs to KYNA, CNQX or APV was not significantly different from that of polysynaptic inputs irrespective of sources of inputs. These results suggest that excitatory synaptic inputs to MVN neurons are mediated mainly through non-NMDA type of EAA receptors from vestibular afferents and through NMDA as well as non-NMDA types of EAA receptors from commissures.     

Involvement of nitric oxide,cyclic GMP and phosphodiesterase 5 in excitatory amino acid and GABA release in the nucleus accumbens evoked by activation of the hippocampal fimbria

It is known that the nucleus accumbens contains all elements of the nitric oxide (NO)-cyclic GMP (cGMP) system but the role of NO in this nucleus is not well understood. We investigated the contribution of the NO-cGMP system in the neurotransmission elicited by hippocampal nerve signals which are propagated to the nucleus accumbens via the fornix/fimbria. This glutamatergic hippocampus-accumbens projection was electrically stimulated for short periods in the urethane-anaesthetized rat. The nucleus accumbens was simultaneously superfused by the push-pull technique with compounds that influence the NO system and the released glutamate, aspartate and GABA were determined in the superfusate.Superfusion of the nucleus accumbens with the NO donor, PAPA/NO, enhanced basal release of the investigated amino acids with a complex concentration dependency. The release of glutamate and aspartate was also increased by the inhibitor of phosphodiesterase 5, UK-114,542. The PAPA/NO-elicited release of glutamate and aspartate was diminished by superfusion with the inhibitor of guanylyl cyclase, NS 2028. Basal release of amino acid transmitters was not influenced by NS 2028 and the NO synthase inhibitor, 7-NINA.Electrical stimulation of the fornix/fimbria increased the outflow of aspartate, glutamate and GABA in the nucleus accumbens. The stimulation-evoked release was abolished by superfusion of the nucleus with tetrodotoxin and strongly diminished by NS 2028, 7-NINA and N(G)-nitro-L-arginine methyl ester (L-name), while PAPA/NO facilitated stimulation-evoked release of these neurotransmitters. UK-114,542 also enhanced the evoked release of glutamate and aspartate while evoked GABA release was not influenced by the phosphodiesterase inhibitor.These findings indicate that NO plays the role of an excitatory transmitter in the nucleus accumbens and that nerve signals from the hippocampus propagated via fornix/fimbria induce NO synthesis in the nucleus accumbens. NO does not exert a tonic influence on basal release but facilitates release of aspartate, glutamate and GABA through increased cGMP synthesis. Phosphodiesterase 5 seems to be involved in the termination of the NO effect in glutamatergic but not in GABAergic neurons.     

An excitatory input to nucleus raphe magnus from the red nucleus in the cat

In chloralose-anaesthetized cats, with the cerebellum removed, stimulation in the red nucleus excited the majority (60-65%) of neurones in nucleus raphe magnus (NRM), including raphespinal neurones. Evidence was obtained for both monosynaptic and polysynaptic excitation. The projection was confirmed by recording antidromic responses in the red nucleus to stimulation in NRM. It is suggested that the role of NRM in motor control is to inhibit spinal flexion responses to peripheral stimuli so that commands from the red nucleus and other motor control regions may take place without interruption.     

Slow excitatory amino acid receptor-mediated synaptic transmission in turtle cerebellar Purkinje cells

1. The excitatory synaptic responses of turtle Purkinje cells to climbing and parallel fiber (CF and PF) stimulation have been studied by the use of intrasomatic and intradendritic recordings in intact cerebellum and brain stem-cerebellum preparations in vitro. 2. Activation of CF inputs from the cerebellar peduncle or the region of the inferior olive evoked complex spikes followed by slow excitatory postsynaptic potentials (EPSPs), both of which were evoked in an all-or-none fashion. 3. Single stimuli applied to the cerebellar molecular layer activated fast PF-mediated EPSPs; brief trains of PF stimuli (2-5 stimuli, 50-100 Hz) evoked volleys of fast EPSPs followed by a slow, long-lasting EPSP. The amplitude of the fast and slow PF-mediated EPSPs were both graded with stimulus intensity. 4. Slow EPSPs evoked both by CF and PF stimulation were associated with an increase in membrane conductance and were increased in amplitude by hyperpolarization. 5. The CF-evoked slow EPSP was profoundly attenuated by repetitive activation at interstimulus intervals of less than 15-20 s, whereas the PF-evoked slow EPSP was not reduced by repetitive activation. 6. The PF-evoked slow EPSP readily triggered dendritic pacemaker discharges when activated at or near resting membrane potential. The activation of this potential by phasic PF volleys may, therefore, provide an appropriate synaptic drive to cerebellar Purkinje cells to entrain the intrinsic pacemaker properties of these cells to cycles of motor activity. 7. Both slow synaptic potentials were blocked by the excitatory amino acid antagonists kynurenate and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not by DL-2-amino-5-phosphonovalerate (DL-AP5) or L-serine-O-phosphate (L-SOP). The PF-evoked slow EPSP was selectively antagonized by L-2-amino-4-phosphonobutyrate (L-AP4; 20-100 microM). 8. It is suggested that the CF- and PF-evoked slow EPSPs observed in this study represent a novel class of excitatory amino acid receptor-mediated slow synaptic potentials activated by Purkinje cell afferents, which may play a role in synaptic integration and motor pattern generation in the cerebellum.