Matrix metalloproteinase‐9 reversibly affects the time course of NMDA‐induced currents in cultured rat hippocampal neurons

Matrix Metalloproteinase 9 (MMP‐9) has been demonstrated to play a crucial role in maintenance of NMDA receptor‐dependent LTP and in lateral mobility of these receptors. However, the effect of MMP‐9 on NMDA receptor (NMDAR) functional properties is unknown. For this purpose we have investigated the impact of recombinant MMP‐9 on the whole‐cell NMDAR‐mediated current responses in cultured hippocampal neurons. Treatment with MMP‐9 induced a reversible acceleration of desensitization and deactivation kinetics but had no effect on current amplitude. Interestingly, phorbol ester, a PKC activator known to enhance NMDAR lateral mobility, induced kinetic changes of currents similar to those produced by MMP‐9. In conclusion, our results show that MMP‐9 reversibly modulates the NMDAR kinetics and raise a possibility that this modulation could be related to the lateral mobility of these receptors. © 2009 Wiley‐Liss, Inc.     

Microglial expression of alphavbeta3 and alphavbeta5 integrins is regulated by cytokines and the extracellular matrix: beta5 integrin null microglia show no defects in adhesion or MMP-9 expression on vitronectin

As the primary immune effector cells in the CNS, microglia play a central role in regulating inflammation. The extracellular matrix (ECM) protein vitronectin is a strong inducer of microglial activation, switching microglia from a resting into an activated potentially destructive phenotype. As the activating effect of vitronectin is mediated by alphav integrins, the aim of the current study was to evaluate the requirement of the alphavbeta5 integrin in mediating microglial adhesion and activation to vitronectin, by studying these events in beta5 integrin-null murine microglia. Surprisingly, beta5 integrin null microglia were not defective in adhesion to vitronectin. Further analysis showed that microglia express the alphavbeta3 integrin, in addition to alphavbeta5. Flow cytometry revealed that microglial alphav integrin expression is regulated by cytokines and ECM proteins. alphavbeta3 integrin expression was downregulated by IFN-gamma, TNF, LPS, and TGF-beta1. alphavbeta5 expression was also reduced by IFN-gamma, TNF, and LPS, but strongly increased by the antiactivating factors TGF-beta1 and laminin. Gel zymography revealed that beta5 integrin null microglia showed no deficiency in their expression of matrix metalloproteinase (MMP)-9 in response to vitronectin. Taken together, these data show that microglia express two different alphav integrins, alphavbeta3 and alphavbeta5, and that expression of these integrins is independently regulated by cytokines and ECM proteins. Furthermore, it reveals that the alphavbeta5 integrin is not essential for mediating microglial adhesion and MMP-9 expression in response to vitronectin.     

Matrix metalloproteinase‐9 expression in the nuclear compartment of neurons and glial cells in aging and stroke

Matrix metalloproteinases (MMPs) are well‐recognized denominators for extracellular matrix remodeling in the pathology of both ischemic and hemorrhagic strokes. Recent data on non‐nervous system tissue showed intracellular and even intranuclear localizations for different MMPs, and together with this, a plethora of new functions have been proposed for these intracellular active enzymes, but are mostly related to apoptosis induction and malign transformation. In neurons and glial cells, on human tissue, animal models and cell cultures, different active MMPs have been also proven to be located in the intra‐cytoplasmic or intra‐nuclear compartments, with no clear‐cut function. In the present study we show for the first time on human tissue the nuclear expression of MMP‐9, mainly in neurons and to a lesser extent in astrocytes. We have studied ischemic and hemorrhagic stroke patients, as well as aged control patients. Age and ischemic suffering seemed to be the best predictors for an elevated MMP‐9 nuclear expression, and there was no evidence of a clear‐cut extracellular proteolytic activity for this compartment, as revealed by intact vascular basement membranes and assessment of vascular densities. More, the majority of the cells expressing MMP‐9 in the nuclear compartment also co‐expressed activated‐caspase 3, indicating a possible link between nuclear MMP‐9 localization and apoptosis in neuronal and glial cells following an ischemic or hemorrhagic event. These results, besides showing for the first time the nuclear localization of MMP‐9 on a large series of human stroke and aged brain tissues, raise new questions regarding the unknown spectrum of the functions MMPs in human CNS pathology.     

Subcellular distribution of low‐voltage activated T‐type Ca2+ channel subunits (Cav3.1 and Cav3.3) in reticular thalamic neurons of the cat

Low‐voltage‐activated (LVA) Ca²⁺ channels play a critical role in the generation of burst firing in the thalamus. Recently, three LVA Ca²⁺ channel isoforms (Ca_v3.1, Ca_v3.2, Ca_v3.3) have been identified in the reticular thalamic nucleus (RE). Previous electrophysiological and modelling studies have suggested that kinetically different T‐type channels might be expressed in a compartmentalized manner in RE cells. However, their precise subcellular distribution has not been fully elucidated. Using light and electron microscopic (EM) immunocytochemistry, we investigated the subcellular expression pattern of Ca_v3.1 and Ca_v3.3 channel subunits in RE neurons of the cat. Fluorescent and peroxidase labelling demonstrated the presence of Ca_v3.1 channel predominantly on the somata and proximal dendrites and Ca_v3.3 channels on cell bodies. Quantitative immunogold localization disclosed that Ca_v3.1 and Ca_v3.3 isoforms showed 5.8‐ and 8.7‐fold higher density, respectively, in the cytoplasm compared with somatic plasma membrane. Density of Ca_v3.1 isoform in the somatic plasma membrane was 2.21‐fold higher compared with Ca_v3.3 subunit. In the dendritic plasma membrane, Ca_v3.1 channel isoform was expressed throughout the entire dendritic tree. In contrast, Ca_v3.3 isoform was absent from large‐caliber, presumably proximal dendritic segments. Quantitative comparison showed that the relative density of immunogold particles compared with dendritic surface was 8.9‐ and 14.8‐fold higher for Ca_v3.1 and Ca_v3.3, respectively, in small‐diameter dendrites than in large proximal dendritic segments or somata. Our results demonstrate a higher density of low‐threshold Ca²⁺ channels in distal dendrites and provide further evidence of the role of RE neuron dendrites in the generation of prolonged, low‐threshold spike bursts. © 2009 Wiley‐Liss, Inc.     

Microglial activation induced by the alarmin S100B is regulated by poly(ADP‐ribose) polymerase‐1

Brain injury resulting from stroke or trauma can be exacerbated by the release of proinflammatory cytokines, proteases, and reactive oxygen species by activated microglia. The microglial activation resulting from brain injury is mediated in part by alarmins, which are signaling molecules released from damaged cells. The nuclear enzyme poly(ADP‐ribose) polymerase‐1 (PARP‐1) has been shown to regulate microglial activation after brain injury, and here we show that signaling effects of the alarmin S100B are regulated by PARP‐1. S100B is a protein localized predominantly to astrocytes. Exogenous S100B added to primary microglial cultures induced a rapid change in microglial morphology, upregulation of IL‐1β, TNFα, and iNOS gene expression, and release of matrix metalloproteinase 9 and nitric oxide. Most, though not all of these effects were attenuated in PARP‐1^‐/‐ microglia and in wild‐type microglia treated with the PARP inhibitor, veliparib. Microglial activation and gene expression changes induced by S100B injected directly into brain were likewise attenuated by PARP‐1 inhibition. The anti‐inflammatory effects of PARP‐1 inhibitors in acutely injured brain may thus be mediated in part through effects on S100B signaling pathways. GLIA 2016;64:1869–1878     

Role of VEGF and matrix metalloproteinase‐9 in peritumoral brain edema associated with supratentorial benign meningiomas

Accumulating evidence indicates that VEGF and matrix metalloproteinase‐9 (MMP‐9) play a central role in the development of peritumoral brain edema (PTBE) associated with human brain tumors. However, the roles of these proteins, particularly of MMP‐9, in PTBE associated with benign meningiomas have not been elucidated. We investigated the association between clinical features and biological factors, such as VEGF and MMP‐9, and the incidence of PTBE and edema index (EI) in 60 patients with benign meningiomas. In this study, supratentorial lesions were examined for evaluating the extent of PTBE in the surrounding normal brain tissue. VEGF and MMP‐9 expression was immunohistochemically examined. Multivariate analysis revealed that the presence of pial blood supply (odds ratio [OR] 12.250; P = 0.0096) and VEGF (OR 7.683; P = 0.0155), but not MMP‐9 (OR 1.178; P = 0.8113), expression are significant factors that independently predict the incidence of PTBE and influence EI. VEGF (P = 0.0397) and MMP‐9 (P = 0.0057) expression correlates with the presence of pial blood supply. Moreover, tumors with high VEGF and MMP‐9 expression had higher EIs than those with high expression of either (P = 0.030). Our findings suggest that MMP‐9 expression was positively related to VEGF expression and pial blood supply and promoted the occurrence of PTBE by inducing the disruption of the arachnoid membrane and formation of pial blood supply.     

Ultrastructural localization of cannabinoid CB1 and mGluR5 receptors in the prefrontal cortex and amygdala

Stimulation of the postsynaptic metabotropic glutamate receptor mGluR5 triggers retrograde signaling of endocannabinoids that activate presynaptic cannabinoid CB1 receptors on juxtaposing axon terminals. To better understand the synaptic structure that supports mGluR5 mediation of CB1 activation in the prefrontal cortex (PFC) and basolateral amygdala (BLA), we examined electron microscopic dual immunolabeling of these receptors in the prelimbic PFC (prPFC) and BLA of adult male rats. CB1 immunoreactivity was detected in axon terminals that were typically large, complex, and contained dense-core and clear synaptic vesicles. Of terminals forming discernible synaptic specializations, 95% were symmetric inhibitory-type in the prPFC and 90% were inhibitory in the BLA. CB1-immunoreactive terminals frequently contacted dendrites containing mGluR5 adjacent to unlabeled terminals forming excitatory-type synapses. Because most CB1-containing terminals form inhibitory-type synapses, the unlabeled axon terminals forming asymmetric synapses are the likely source of the mGluR5 ligand glutamate. In the prPFC, serial section analysis revealed that GABAergic CB1-containing axon terminals targeted dendrites adjacent to glutamatergic axon terminals, often near dendritic bifurcations. These observations provide ultrastructural evidence that cortical CB1 receptors are strategically positioned for integration of synaptic signaling in response to stimulation of postsynaptic mGluR5 receptors and facilitation of heterosynaptic communication between multiple neurons.     

Subcellular distribution of α1G subunit of T‐type calcium channel in the mouse dorsal lateral geniculate nucleus

T‐type calcium channels play a pivotal role in regulating neural membrane excitability in the nervous system. However, the precise subcellular distributions of T‐type channel subunits and their implication for membrane excitability are not well understood. Here we investigated the subcellular distribution of the α1G subunit of the calcium channel which is expressed highly in the mouse dorsal lateral geniculate nucleus (dLGN). Light microscopic analysis demonstrated that dLGN exhibits intense immunoperoxidase reactivity for the α1G subunit. Electron microscopic observation showed that the labeling was present in both the relay cells and interneurons and was found in the somatodendritic, but not axonal, domains of these cells. Most of the immunogold particles for the α1G subunit were either associated with the plasma membrane or the intracellular membranes. Reconstruction analysis of serial electron microscopic images revealed that the intensity of the intracellular labeling exhibited a gradient such that the labeling density was higher in the proximal dendrite and progressively decreased towards the distal dendrite. In contrast, the plasma membrane‐associated particles were distributed with a uniform density over the somatodendritic surface of dLGN cells. The labeling density in the relay cell plasma membrane was about 3‐fold higher than that of the interneurons. These results provide ultrastructural evidence for cell‐type‐specific expression levels and for uniform expression density of the α1G subunit over the plasma membrane of dLGN cells. J. Comp. Neurol. 518:4362–4374, 2010. © 2010 Wiley‐Liss, Inc.     

Fluoxetine‐induced plasticity in the visual cortex outlasts the duration of the naturally occurring critical period

Heightened neuronal plasticity expressed during early postnatal life has been thought to permanently decline once critical periods have ended. For example, monocular deprivation is able to shift ocular dominance in the mouse visual cortex during the first months of life, but this effect is lost later in life. However, various treatments, such as the antidepressant fluoxetine, can reactivate a critical period‐like plasticity in the adult brain. When monocular deprivation is supplemented with chronic fluoxetine administration, a major shift in ocular dominance is produced after the critical period has ended. In the current study, we characterized the temporal patterns of fluoxetine‐induced plasticity in the adult mouse visual cortex, using in vivo optical imaging. We found that artificially induced plasticity in ocular dominance extended beyond the duration of the naturally occurring critical period and continued as long as fluoxetine was administered. However, this fluoxetine‐induced plasticity period ended as soon as the drug was not given. These features of antidepressant‐induced plasticity may be useful when designing treatment strategies involving long‐term antidepressant treatment in humans.     

Subcellular targeting of kappa‐opioid receptors in the rat nucleus locus coeruleus

The dynorphin (DYN)‐kappa opioid receptor (κOR) system has been implicated in stress modulation, depression, and relapse to drug‐seeking behaviors. Previous anatomical and physiological data have indicated that the noradrenergic nucleus locus coeruleus (LC) is one site at which DYN may contribute to these effects. Using light microscopy, immunofluorescence, and electron microscopy, the present study investigated the cellular substrates for pre‐ and postsynaptic interactions of κOR in the LC. Dual immunocytochemical labeling for κOR and tyrosine hydroxylase (TH) or κOR and preprodynorphin (ppDYN) was examined in the same section of tissue. Light microscopic analysis revealed prominent κOR immunoreactivity in the nuclear core of the LC and in the peri‐coerulear region where noradrenergic dendrites extend. Fluorescence and electron microscopy revealed κOR immunoreactivity within TH‐immunoreactive somata and dendrites in the LC as well as localized to ppDYN‐immunoreactive processes. In sections processed for κOR and TH, ≈29% (200/688) of the κOR‐containing axon terminals identified targeted TH‐containing profiles. Approximately 49% (98/200) of the κOR‐labeled axon terminals formed asymmetric synapses with TH‐labeled dendrites. Sections processed for κOR and ppDYN showed that, of the axon terminals exhibiting κOR, 47% (223/477) also exhibited ppDYN. These findings indicate that κORs are poised to modulate LC activity by their localization to somata and dendrites. Furthermore, κORs are strategically localized to presynaptically modulate DYN afferent input to catecholamine‐containing neurons in the LC. These data add to the growing literature showing that κORs can modulate diverse afferent signaling to the LC. J. Comp. Neurol. 512:419–431, 2009. © 2008 Wiley‐Liss, Inc.     

The first thalamocortical synapses are made in the cortical plate in the developing visual cortex of the wallaby (Macropus eugenii)

The time course of development and laminar distribution of thalamocortical synapses in the visual cortex of the marsupial mammal the wallaby (Macropus eugenii) has been studied by electron microscopy from the time of afferent ingrowth to the appearance of layer 4, the main target for thalamic axons. Axons were labeled from the thalamus by a fluorescent carbocyanine dye in fixed tissue or by transneuronal transport of horseradish peroxidase conjugated to wheat germ agglutinin from the eye. Thalamic axons first reached the cortex 2 weeks after birth and grew into the developing cortical plate without a waiting period in the subplate. The first thalamocortical synapses were detected 2 weeks later solely throughout the loosely packed zone of the cortical plate, where layer 6 cells previously have been shown to reside. As the thickness of the cortex increased with age, thalamocortical synapses were increasingly prevalent in the loosely packed zone of the cortical plate. With the appearance of layer 4, thalamocortical synapses were found there as well as in the marginal zone and layer 6. There was no evidence for an early population of thalamocortical synapses in the subplate. The first synapses made by thalamic axons were in a region containing layer 6 cells, one of their normal targets in the mature cortex.     

Direction selectivity of neurons in the visual cortex is non‐linear and lamina‐dependent

Neurons in the visual cortex are generally selective to direction of movement of a stimulus. Although models of this direction selectivity (DS) assume linearity, experimental data show stronger degrees of DS than those predicted by linear models. Our current study was intended to determine the degree of non‐linearity of the DS mechanism for cells within different laminae of the cat's primary visual cortex. To do this, we analysed cells in our database by using neurophysiological and histological approaches to quantify non‐linear components of DS in four principal cortical laminae (layers 2/3, 4, 5, and 6). We used a DS index (DSI) to quantify degrees of DS in our sample. Our results showed laminar differences. In layer 4, the main thalamic input region, most neurons were of the simple type and showed high DSI values. For complex cells in layer 4, there was a broad distribution of DSI values. Similar features were observed in layer 2/3, but complex cells were dominant. In deeper layers (5 and 6), DSI value distributions were characterized by clear peaks at high values. Independently of specific lamina, high DSI values were accompanied by narrow orientation tuning widths. Differences in orientation tuning for non‐preferred vs. preferred directions were smallest in layer 4 and largest in layer 6. These results are consistent with a non‐linear process of intra‐cortical inhibition that enhances DS by selective suppression of neuronal firing for non‐preferred directions of stimulus motion in a lamina‐dependent manner. Other potential mechanisms are also considered.     

Increased 5‐HT2A receptors in the temporal cortex of parkinsonian patients with visual hallucinations

Well‐formed visual hallucinations (VH) are common in patients with Parkinson's disease (PD). The pathophysiology of VH in PD is unknown but may involve structures mediating visual processing such as the inferior temporal cortex. Serotonergic type 2A (5‐HT_2A) receptors have been linked to many psychiatric disorders, including psychosis. We hypothesized that enhanced 5‐HT_2A receptor levels may be involved in VH in PD. Autoradiographic binding using [³H]‐ketanserin and spiperone, to define 5‐HT_2A receptors, was performed in 6 PD patients with VH, 6 PD patients without VH, and 5 healthy, age‐matched controls. The cerebral regions studied included the orbitofrontal cortex, inferolateral temporal cortex, motor cortex, striatum, and substantia nigra. There was a significant (45.6%) increase in the levels of [³H]‐ketanserin binding in the inferolateral temporal cortex of PD patients with VH when compared with PD patients without VH (54.3 ± 5.2 fmol/mg vs. 37.3 ± 4.3 fmol/mg, P = 0.039). Additionally, there was a significant increase in the levels of 5‐HT_2A receptors in the motor cortex of all PD patients taken as a group when compared with controls (57.8 ± 5.7 fmol/mg vs. 41.2 ± 2.6 fmol/mg, P = 0.0297). These results suggest that enhanced 5‐HT_2A‐mediated neurotransmission in the inferolateral temporal cortex, a critical structure in visual processing, might be associated with the development of VH in PD. Our results provide new insights into the pathophysiology of VH in PD and provide an anatomical basis to explain why compounds with 5‐HT_2A antagonist activity are effective at alleviating this debilitating complication. © 2010 Movement Disorder Society     

SPARC-like 1 (SC1) is a diversely expressed and developmentally regulated matricellular protein that does not compensate for the absence of SPARC in the CNS

SPARC-like 1 (SC1) is a member of the SPARC family of matricellular proteins that has been implicated in the regulation of processes such as cell migration, proliferation, and differentiation. Here we show that SC1 exhibits remarkably diverse and dynamic expression in the developing and adult nervous system. During development, SC1 localizes to radial glia and pial-derived structures, including the vasculature, choroid plexus, and pial membranes. SC1 is not downregulated in postnatal development, but its expression shifts to distinct time windows in subtypes of glia and neurons, including astrocytes, large projection neurons, Bergmann glia, Schwann cells, and ganglionic satellite cells. In addition, SC1 expression levels and patterns are not altered in the SPARC null mouse, suggesting that SC1 does not compensate for the absence of SPARC. We conclude that SC1 and SPARC may share significant homology, but are likely to have distinct but complementary roles in nervous system development.     

Acute effect of carbamazepine on corticothalamic 5–9‐Hz and thalamocortical spindle (10–16‐Hz) oscillations in the rat

A major side effect of carbamazepine (CBZ), a drug used to treat neurological and neuropsychiatric disorders, is drowsiness, a state characterized by increased slow‐wave oscillations with the emergence of sleep spindles in the electroencephalogram (EEG). We conducted cortical EEG and thalamic cellular recordings in freely moving or lightly anesthetized rats to explore the impact of CBZ within the intact corticothalamic (CT)–thalamocortical (TC) network, more specifically on CT 5–9‐Hz and TC spindle (10–16‐Hz) oscillations. Two to three successive 5–9‐Hz waves were followed by a spindle in the cortical EEG. A single systemic injection of CBZ (20 mg/kg) induced a significant increase in the power of EEG 5–9‐Hz oscillations and spindles. Intracellular recordings of glutamatergic TC neurons revealed 5–9‐Hz depolarizing wave–hyperpolarizing wave sequences prolonged by robust, rhythmic spindle‐frequency hyperpolarizing waves. This hybrid sequence occurred during a slow hyperpolarizing trough, and was at least 10 times more frequent under the CBZ condition than under the control condition. The hyperpolarizing waves reversed at approximately ?70 mV, and became depolarizing when recorded with KCl‐filled intracellular micropipettes, indicating that they were GABA_A receptor‐mediated potentials. In neurons of the GABAergic thalamic reticular nucleus, the principal source of TC GABAergic inputs, CBZ augmented both the number and the duration of sequences of rhythmic spindle‐frequency bursts of action potentials. This indicates that these GABAergic neurons are responsible for the generation of at least the spindle‐frequency hyperpolarizing waves in TC neurons. In conclusion, CBZ potentiates GABA_A receptor‐mediated TC spindle oscillations. Furthermore, we propose that CT 5–9‐Hz waves can trigger TC spindles.     

Activation of β‐adrenergic receptors in rat visual cortex expands astrocytic processes and reduces extracellular space volume

Brain extracellular space (ECS) is an interconnected channel that allows diffusion‐mediated transport of signaling molecules, metabolites, and drugs. We tested the hypothesis that β‐adrenergic receptor (βAR) activation impacts extracellular diffusion‐mediated transport of molecules through alterations in the morphology of astrocytes. Two structural parameters of ECS—volume fraction and tortuosity—govern extracellular diffusion. Volume fraction (α) is the volume of ECS relative to the total tissue volume. Tortuosity (λ) is a measure of the hindrance that molecules experience in the ECS, compared to a free medium. The real‐time iontophoretic (RTI) method revealed that treatment of acutely prepared visual cortical slices of adult female rats with a βAR agonist, DL‐isoproterenol (ISO), decreases α significantly, from 0.22 ± 0.03 (mean ± SD) for controls without agonist to 0.18 ± 0.03 with ISO, without altering λ (control: 1.64 ± 0.04; ISO: 1.63 ± 0.04). Electron microscopy revealed that the ISO treatment significantly increased the cytoplasmic area of astrocytic distal endings per unit area of neuropil by 54%. These findings show that norepinephrine decreases α, in part, through an increase in astrocytic volume following βAR activation. Norepinephrine is recognized to be released within the brain during the awake state and increase neurons’ signal‐to‐noise ratio through modulation of neurons’ biophysical properties. Our findings uncover a new mechanism for noradrenergic modulation of neuronal signals. Through astrocytic activation leading to a reduction of α, noradrenergic modulation increases extracellular concentration of neurotransmitters and neuromodulators, thereby facilitating neuronal interactions, especially during wakefulness. Synapse 70:307–316, 2016 . © 2016 Wiley Periodicals, Inc.     

Subcellular distribution of 5-HT(1B) and 5-HT(7) receptors in the mouse suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN), a circadian oscillator, receives glutamatergic afferents from the retina and serotonergic (5-HT) afferents from the median raphe. 5-HT(1B) and 5-HT(7) receptor agonists inhibit the effects of light on SCN circadian activity. Electron microscopic (EM) immunocytochemical procedures were used to determine the subcellular localization of 5-HT(1B) and 5-HT(7) receptors in the SCN. 5-HT(1B) receptor immunostaining was associated with the plasma membrane of thin unmyelinated axons, preterminal axons, and terminals of optic and nonoptic origin. 5-HT(1B) receptor immunostaining in terminals was almost never observed at the synaptic active zone. To a much lesser extent, 5-HT(1B) immunoreaction product was noted in dendrites and somata of SCN neurons. 5-HT(7) receptor immunoreactivity in gamma-aminobutyric acid (GABA), vasoactive intestinal polypeptide (VIP), and vasopressin (VP) neuronal elements in the SCN was examined by using double-label procedures. 5-HT(7) receptor immunoreaction product was often observed in GABA-, VIP-, and VP-immunoreactive dendrites as postsynaptic receptors and in axonal terminals as presynaptic receptors. 5-HT(7) receptor immunoreactivity in terminals and dendrites was often associated with the plasma membrane but very seldom at the active zone. In GABA-, VIP-, and VP-immunoreactive perikarya, 5-HT(7) receptor immunoreaction product was distributed throughout the cytoplasm often in association with the endoplasmic reticulum and the Golgi complex. The distribution of 5-HT(1B) receptors in presynaptic afferent terminals and postsynaptic SCN processes, as well as the distribution of 5-HT(7) receptors in both pre- and postsynaptic GABA, VIP, and VP SCN processes, suggests that serotonin plays a significant role in the regulation of circadian rhythms by modulating SCN synaptic activity.