Mechanisms Of Synaptic Transmission Research Articles

Local field potential journey into the Basal Ganglia

Local field potentials (LFP) in the basal ganglia (BG) have attracted considerable research and clinical interest. The genesis of these signals has been a topic of extensive discourse, focusing on whether they are a manifestation of local synaptic activity or result from the propagation of electrical signals through tissue, as described by the Maxwell equations (volume conduction). To investigate this, we conducted simultaneous recordings of LFPs from two cortical areas— the dorsolateral prefrontal cortex (DLPFC) and the primary motor cortex (M1)—and various sites within the BG nuclei in an awake, non-task-engaged non-human primate (NHP). Employing innovative analytical techniques, we discerned significant cross-correlations indicative of potential connections, while filtering out non-significant correlations. This allowed us to differentiate between synaptic inputs and volume conduction. Our findings indicate two distinct propagation pathways of BG field potentials emanating from the M1 and the DLPFC, each characterized by different temporal delays. The results imply that these anatomical pathways are differentially influenced by the mechanisms of volume conduction and synaptic transmission. Notably, the M1 exhibits more functional links with non-zero-time delays to the BG structures, while the DLPFC-BG connections are marked by zero-time delays, suggesting a predominance of volume conduction effects. Consequently, investigations into the origins of BG LFP should account for the distinct anatomical pathways linking the cortex and the BG, as they differentially represent information flow and volume conductance.

Synaptic Electrophysiology of the Drosophila Neuromuscular Junction.

Chemical synaptic transmission is an important means of neuronal communication in the nervous system. Upon the arrival of an action potential, the nerve terminal experiences an influx of calcium ions, which in turn trigger the exocytosis of synaptic vesicles (SVs) and the release of neurotransmitters into the synaptic cleft. Transmitters elicit synaptic responses in the postsynaptic cell by binding to and activating specific receptors. This is followed by the recycling of SVs at presynaptic terminals. The Drosophila larval neuromuscular junction (NMJ) shares many structural and functional similarities to synapses in other animals, including humans. These include the basic features of synaptic transmission, as well as the molecular mechanisms regulating the SV cycle. Because of its large size, easy accessibility, and well-characterized genetics, the fly NMJ is an excellent model system for dissecting the cellular and molecular mechanisms of synaptic transmission. Here, we describe the theory and practice of electrophysiology as applied to the Drosophila larval NMJ preparation. We introduce the basics of membrane potentials, with an emphasis on the resting potential and synaptic potential. We also describe the equipment and methods required to set up an electrophysiology rig.

Structure, biophysics, and circuit function of a "giant" cortical presynaptic terminal.

The hippocampal mossy fiber synapse, formed between axons of dentate gyrus granule cells and dendrites of CA3 pyramidal neurons, is a key synapse in the trisynaptic circuitry of the hippocampus. Because of its comparatively large size, this synapse is accessible to direct presynaptic recording, allowing a rigorous investigation of the biophysical mechanisms of synaptic transmission and plasticity. Furthermore, because of its placement in the very center of the hippocampal memory circuit, this synapse seems to be critically involved in several higher network functions, such as learning, memory, pattern separation, and pattern completion. Recent work based on new technologies in both nanoanatomy and nanophysiology, including presynaptic patch-clamp recording, paired recording, super-resolution light microscopy, and freeze-fracture and "flash-and-freeze" electron microscopy, has provided new insights into the structure, biophysics, and network function of this intriguing synapse. This brings us one step closer to answering a fundamental question in neuroscience: how basic synaptic properties shape higher network computations.

Effect of terahertz waves on the aggregation behavior of neurotransmitters.

Understanding the dynamics of neurotransmitters is crucial for unraveling synaptic transmission mechanisms in neuroscience. In this study, we investigated the impact of terahertz (THz) waves on the aggregation of four common neurotransmitters through all-atom molecular dynamics (MD) simulations. The simulations revealed enhanced nicotine (NCT) aggregation under 11.05 and 21.44 THz, with a minimal effect at 42.55 THz. Structural analysis further indicated strengthened intermolecular interactions and weakened hydration effects under specific THz stimulation. In addition, enhanced aggregation was observed at stronger field strengths, particularly at 21.44 THz. Furthermore, similar investigations on epinephrine (EPI), 5-hydroxytryptamine (5-HT), and γ-aminobutyric acid (GABA) corroborated these findings. Notably, EPI showed increased aggregation at 19.05 THz, emphasizing the influence of vibrational modes on aggregation. However, 5-HT and GABA, with charged or hydrophilic functional groups, exhibited minimal aggregation under THz stimulation. The present study sheds some light on neurotransmitter responses to THz waves, offering implications for neuroscience and interdisciplinary applications.

Picroside Ⅱ attenuates neuropathic pain by regulating inflammation and spinal excitatory synaptic transmission.

Nerve injury induced microglia activation, which released inflammatory mediators and developed neuropathic pain. Picroside Ⅱ (PⅡ) attenuated neuropathic pain by inhibiting the neuroinflammation of the spinal dorsal horn; however, how it engaged in the cross talk between microglia and neurons remained ambiguous. This study aimed to investigate PⅡ in the modulation of spinal synaptic transmission mechanisms on pain hypersensitivity in neuropathic rats. We investigated the analgesia of PⅡ in mechanical and thermal hyperalgesia using the spinal nerve ligation (SNL)-induced neuropathic pain model and formalin-induced tonic pain model, respectively. RNA sequencing and network pharmacology were employed to screen core targets and signaling pathways. Immunofluorescence staining and qPCR were performed to explore the expression level of microglia and inflammatory mediator mRNA. The whole-cell patch-clamp recordings were utilized to record miniature excitatory postsynaptic currents in excitatory synaptic transmission. Our results demonstrated that the analgesic of PⅡ was significant in both pain models, and the underlying mechanism may involve inflammatory signaling pathways. PⅡ reversed the SNL-induced overexpression of microglia and inflammatory factors. Moreover, PⅡ dose dependently inhibited excessive glutamate transmission. Thus, this study suggested that PⅡ attenuated neuropathic pain by inhibiting excitatory glutamate transmission of spinal synapses, induced by an inflammatory response on microglia.

Staying Up to Date With Evolving Postpartum Depression Pathophysiology and Treatment Research.

Perinatal depression (PND) is one of the most common medical conditions associated with pregnancy, with 1 in 7 women impacted by PND symptoms and 1 in 13 meeting criteria for major depressive disorder. Unfortunately, half of postpartum depression (PPD) cases begin during pregnancy but are not diagnosed until postpartum. Delayed diagnosis and treatment of PND lead to poor outcomes for both mother and child. The American College of Obstetricians and Gynecologists recently updated its recommendation that screening for perinatal depression and anxiety occur at the initial prenatal visit, later in pregnancy, and at postpartum. Several hypotheses have been developed to explain the pathophysiology of PND including endocrine, epigenetic, synaptic transmission, neural network, neurosteroid, stress, and inflammatory mechanisms. Researchers believe that the answer lies in a synthesized mechanism of all of these models. Novel and emerging therapeutics are focusing on the neurosteroid mechanism within the integrated hypothesis. Neuroactive steroids are changing the understanding of the pathophysiology of depression and PPD, and novel and emerging therapeutics with new mechanisms of action based on these findings are impacting the treatment paradigm for this widespread and burdensome disorder.

Age-Dependent Behavioral and Synaptic Dysfunction Impairment Are Improved with Long-Term Andrographolide Administration in Long-Lived Female Degus (Octodon degus).

In Octodon degus, the aging process is not equivalent between sexes and worsens for females. To determine the beginning of detrimental features in females and the ways in which to improve them, we compared adult females (36 months old) and aged females (72 months old) treated with Andrographolide (ANDRO), the primary ingredient in Andrographis paniculata. Our behavioral data demonstrated that age does not affect recognition memory and preference for novel experiences, but ANDRO increases these at both ages. Sociability was also not affected by age; however, social recognition and long-term memory were lower in the aged females than adults but were restored with ANDRO. The synaptic physiology data from brain slices showed that adults have more basal synaptic efficiency than aged degus; however, ANDRO reduced basal activity in adults, while it increased long-term potentiation (LTP). Instead, ANDRO increased the basal synaptic activity and LTP in aged females. Age-dependent changes were also observed in synaptic proteins, where aged females have higher synaptotagmin (SYT) and lower postsynaptic density protein-95 (PSD95) levels than adults. ANDRO increased the N-methyl D-aspartate receptor subtype 2B (NR2B) at both ages and the PSD95 and Homer1 only in the aged. Thus, females exposed to long-term ANDRO administration show improved complex behaviors related to age-detrimental effects, modulating mechanisms of synaptic transmission, and proteins.

Local brain environment changes associated with epileptogenesis.

Plastic change of the neuronal system has traditionally been assumed to be governed primarily by the long-term potentiation/depression mechanisms of synaptic transmission. However, a rather simple shift in the ambient ion, transmitter and metabolite concentrations could have a pivotal role in generating plasticity upon the physiological process of learning and memory. Local brain environment and metabolic changes could also be the cause and consequences of the pathogenesis leading to epilepsy. Governing of the local brain environment is the primal function of astrocytes. The metabolic state of the entire brain is strongly linked to the activity of the lateral hypothalamus. In this study, plastic change of astrocyte reactions in the lateral hypothalamus was examined using epileptogenesis as an extreme form of plasticity. Fluorescent sensors for calcium or pH expressed in astrocytes were examined for up to one week by in vivo fibre photometry in freely moving transgenic male mice. Optical fluctuations on a timescale of seconds is difficult to assess because these signals are heavily influenced by local brain blood volume changes and pH changes. Using a newly devised method for the analysis of the optical signals, changes in Ca2+ and pH in astrocytes and changes in local brain blood volume associated with hippocampal-stimulated epileptic seizures were extracted. Following a transient alkaline shift in the astrocyte triggered by neuronal hyperactivity, a prominent acidic shift appeared in response to intensified seizure which developed with kindling. The acidic shift was unexpected as transient increase in local brain blood volume was observed in response to intensified seizures, which should lead to efficient extrusion of the acidic CO2. The acidic shift could be a result of glutamate transporter activity and/or due to the increased metabolic load of astrocytes leading to increased CO2 and lactate production. This acidic shift may trigger additional gliotransmitter release from astrocytes leading to the exacerbation of epilepsy. As all cellular enzymic reactions are influenced by Ca2+ and pH, changes in these parameters could also have an impact on the neuronal circuit activity. Thus, controlling the astrocyte pH and/or Ca2+ could be a new therapeutic target for treatment of epilepsy or prevention of undesired plasticity associated with epileptogenesis.

Revisiting the molecular basis of synaptic transmission.

Measurements of the time elapsed during synaptic transmission has shown that synaptic vesicle (SV) fusion lags behind Ca2+-influx by approximately 60 microseconds (µsec). The conventional model cannot explain this extreme rapidity of the release event. Synaptic transmission occurs at the active zone (AZ), which comprises of two pools of SV, non-releasable "tethered" vesicles, and a readily-releasable pool of channel-associated Ca2+-primed vesicles, "RRP". A recent TIRF study at cerebellar-mossy fiber-terminal, showed that subsequent to an action potential, newly "tethered" vesicles, became fusion-competent in a Ca2+-dependent manner, 300-400ms after tethering, but were not fused. This time resolution may correspond to priming of tethered vesicles through Ca2+-binding to Syt1/Munc13-1/complexin. It confirms that Ca2+-priming and Ca2+-influx-independent fusion, are two distinct events. Notably, we have established that Ca2+ channel signals evoked-release in an ion flux-independent manner, demonstrated by Ca2+-impermeable channel, or by substitution of Ca2+ with channel -impermeable La3+. Thus, conformational changes in a channel coupled to RRP appear to directly activate the release machinery and account for a µsec Ca2+-influx-independent vesicle fusion. Rapid vesicle fusion driven by non-ionotropic channel signaling strengthens a conformational-coupling mechanism of synaptic transmission, and contributes to better understanding of neuronal communication vital for brain function.

Purkinje Cell Activity Resonation Generates Rhythmic Behaviors at the Preferred Frequency of 8 Hz.

Neural activity exhibits oscillations, bursts, and resonance, enhancing responsiveness at preferential frequencies. For example, theta-frequency bursting and resonance in granule cells facilitate synaptic transmission and plasticity mechanisms at the input stage of the cerebellar cortex. However, whether theta-frequency bursting of Purkinje cells is involved in generating rhythmic behavior has remained neglected. We recorded and optogenetically modulated the simple and complex spike activity of Purkinje cells while monitoring whisker movements with a high-speed camera of awake, head-fixed mice. During spontaneous whisking, both simple spike activity and whisker movement exhibit peaks within the theta band. Eliciting either simple or complex spikes at frequencies ranging from 0.5 to 28 Hz, we found that 8 Hz is the preferred frequency around which the largest movement is induced. Interestingly, oscillatory whisker movements at 8 Hz were also generated when simple spike bursting was induced at 2 and 4 Hz, but never via climbing fiber stimulation. These results indicate that 8 Hz is the resonant frequency at which the cerebellar-whisker circuitry produces rhythmic whisking.

DNA sequence features underlying large-scale duplications and deletions in human

Copy number variants (CNVs) may cover up to 12% of the whole genome and have substantial impact on phenotypes. We used 5867 duplications and 33,181 deletions available from the 1000 Genomes Project to characterise genomic regions vulnerable to CNV formation and to identify sequence features characteristic for those regions. The GC content for deletions was lower and for duplications was higher than for randomly selected regions. In regions flanking deletions and downstream of duplications, content was higher than in the random sequences, but upstream of duplication content was lower. In duplications and downstream of deletion regions, the percentage of low-complexity sequences was not different from the randomised data. In deletions and upstream of CNVs, it was higher, while for downstream of duplications, it was lower as compared to random sequences. The majority of CNVs intersected with genic regions — mainly with introns. GC content may be associated with CNV formation and CNVs, especially duplications are initiated in low-complexity regions. Moreover, CNVs located or overlapped with introns indicate their role in shaping intron variability. Genic CNV regions were enriched in many essential biological processes such as cell adhesion, synaptic transmission, transport, cytoskeleton organization, immune response and metabolic mechanisms, which indicates that these large-scaled variants play important biological roles.

CLC Anion/Proton Exchangers Regulate Secretory Vesicle Filling and Granule Exocytosis in Chromaffin Cells.

ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knock-out animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENT Cl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells; however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.

Butyric Acid Precursor Tributyrin Modulates Hippocampal Synaptic Plasticity and Prevents Spatial Memory Deficits: Role of PPARγ and AMPK.

BackgroundShort chain fatty acids (SCFA), such as butyric acid (BA), derived from the intestinal fermentation of dietary fiber and contained in dairy products, are gaining interest in relation to their possible beneficial effects on neuropsychological disordersMethodsC57BL/6J male mice were used to investigate the effect of tributyrin (TB), a prodrug of BA, on hippocampus (HIP)-dependent spatial memory, HIP synaptic transmission and plasticity mechanisms, and the expression of genes and proteins relevant to HIP glutamatergic transmission.ResultsEx vivo studies, carried out in HIP slices, revealed that TB can transform early-LTP into late-LTP (l-LTP) and to rescue LTP-inhibition induced by scopolamine. The facilitation of l-LTP induced by TB was blocked both by GW9662 (a PPARγ antagonist) and C-Compound (an AMPK inhibitor), suggesting the involvement of both PPARγ and AMPK on TB effects. Moreover, 48-hour intake of a diet containing 1% TB prevented, in adolescent but not in adult mice, scopolamine-induced impairment of HIP-dependent spatial memory. In the adolescent HIP, TB upregulated gene expression levels of Pparg, leptin, and adiponectin receptors, and that of the glutamate receptor subunits AMPA-2, NMDA-1, NMDA-2A, and NMDA-2B.ConclusionsOur study shows that TB has a positive influence on LTP and HIP-dependent spatial memory, which suggests that BA may have beneficial effects on memory.

Exploring Subcellular Cerebellar Fractions with the Electron Microscope.

Differential ultracentrifugation and subcellular fractionation historically helped to study the components of the cell, to discover new cellular organelles, and to decipher their morphological and molecular properties. In neuroscience, the technique has yielded important results on neuron biochemistry and the mechanisms of synaptic transmission. This Cerebellar Classic is devoted to the pioneering work of Manuel del Cerro, Ray S. Snider, and Mary Lou Oster-Granite, who isolated purified fractions after successive centrifugations of the rat cerebellum from birth to adulthood and studied them under the electron microscope.

Spinal microglial β-endorphin signaling mediates IL-10 and exenatide-induced inhibition of synaptic plasticity in neuropathic pain.

AimThis study aimed to investigate the regulation of pain hypersensitivity induced by the spinal synaptic transmission mechanisms underlying interleukin (IL)‐10 and glucagon‐like peptide 1 receptor (GLP‐1R) agonist exenatide‐induced pain anti‐hypersensitivity in neuropathic rats through spinal nerve ligations.MethodsNeuropathic pain model was established by spinal nerve ligation of L5/L6 and verified by electrophysiological recording and immunofluorescence staining. Microglial expression of β‐endorphin through autocrine IL‐10‐ and exenatide‐induced inhibition of glutamatergic transmission were performed by behavioral tests coupled with whole‐cell recording of miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) through application of endogenous and exogenous IL‐10 and β‐endorphin.ResultsIntrathecal injections of IL‐10, exenatide, and the μ‐opioid receptor (MOR) agonists β‐endorphin and DAMGO inhibited thermal hyperalgesia and mechanical allodynia in neuropathic rats. Whole‐cell recordings of bath application of exenatide, IL‐10, and β‐endorphin showed similarly suppressed enhanced frequency and amplitude of the mEPSCs in the spinal dorsal horn neurons of laminae II, but did not reduce the frequency and amplitude of mIPSCs in neuropathic rats. The inhibitory effects of IL‐10 and exenatide on pain hypersensitive behaviors and spinal synaptic plasticity were totally blocked by pretreatment of IL‐10 antibody, β‐endorphin antiserum, and MOR antagonist CTAP. In addition, the microglial metabolic inhibitor minocycline blocked the inhibitory effects of IL‐10 and exenatide but not β‐endorphin on spinal synaptic plasticity.ConclusionThis suggests that spinal microglial expression of β‐endorphin mediates IL‐10‐ and exenatide‐induced inhibition of glutamatergic transmission and pain hypersensitivity via presynaptic and postsynaptic MORs in spinal dorsal horn.

Estrogen pendulum in schizophrenia and Alzheimer’s disease: Review of therapeutic benefits and outstanding questions

Although produced largely in the periphery, gonadal steroids play a key role in regulating the development and functions of the central nervous system and have been implicated in several chronic neuropsychiatric disorders, with schizophrenia and Alzheimer’s disease (AD) most prominent. Despite major differences in pathobiology and clinical manifestations, in both conditions, estrogen transpires primarily with protective effects, buffering the onset and progression of diseases at various levels. As a result, estrogen replacement therapy (ERT) emerges as one of the most widely discussed adjuvant interventions. In this review, we revisit evidence supporting the protective role of estrogen in schizophrenia and AD and consider putative cellular and molecular mechanisms. We explore the underlying functional processes relevant to the manifestation of these devastating conditions, with a focus on synaptic transmission and plasticity mechanisms. We discuss specific effects of estrogen deficit on neurotransmitter systems such as cholinergic, dopaminergic, serotoninergic, and glutamatergic. While the evidence from both, preclinical and clinical reports, in general, are supportive of the protective effects of estrogen from cognitive decline to synaptic pathology, numerous questions remain, calling for further research.

The glutamatergic synapse: a complex machinery for information processing.

Being the most abundant synaptic type, the glutamatergic synapse is responsible for the larger part of the brain's information processing. Despite the conceptual simplicity of the basic mechanism of synaptic transmission, the glutamatergic synapse shows a large variation in the response to the presynaptic release of the neurotransmitter. This variability is observed not only among different synapses but also in the same single synapse. The synaptic response variability is due to several mechanisms of control of the information transferred among the neurons and suggests that the glutamatergic synapse is not a simple bridge for the transfer of information but plays an important role in its elaboration and management. The control of the synaptic information is operated at pre, post, and extrasynaptic sites in a sort of cooperation between the pre and postsynaptic neurons which also involves the activity of other neurons. The interaction between the different mechanisms of control is extremely complicated and its complete functionality is far from being fully understood. The present review, although not exhaustively, is intended to outline the most important of these mechanisms and their complexity, the understanding of which will be among the most intriguing challenges of future neuroscience.

Delineating the mechanisms of synaptic transmission for the development of targeted neurotherapeutics

Delineating the mechanisms of synaptic transmission for the development of targeted neurotherapeutics

Giving names to the actors of synaptic transmission: The long journey from synaptic vesicles to neural plasticity.

More than a scientific paper or a review article, this is a remembrance of a unique time of science and life that the authors spent in Paul Greengard's laboratory at the Rockefeller University in New York in the 1980s and 1990s, forming the so-called synaptic vesicle group. It was a time in which the molecular mechanisms of synaptic transmission and the nature of the organelles in charge of storing and releasing neurotransmitter were just beginning to be understood. It was an exciting time in which the protein composition of synaptic vesicles started to be identified. It turned out that the interactions of synaptic vesicle proteins with the cytoskeleton and the presynaptic membrane and their modulation by protein phosphorylation represented an essential network regulating the efficiency of neurotransmitter release and thereby synaptic strength and plasticity. This is also a description of the distinct scientific journeys that the three authors took on going back to Europe and how they were strongly influenced by the generous and outstanding mentorship of Paul Greengard, his genuine interest in their lives and careers and the life-long friendship with him.

Loss of Dysbindin Implicates Synaptic Vesicle Replenishment Dysregulation as a Potential Pathogenic Mechanism in Schizophrenia

The schizophrenia-susceptibility gene, dystrobrevin-binding protein 1 (DTNBP1), encodes the dysbindin protein and mediates neurotransmission and neurodevelopment in normal subjects. Functional studies show that DTNBP1 loss may cause deficient presynaptic vesicle transmission, which is related to multiple psychiatric disorders. However, the functional mechanism of dysbindin-mediated synaptic vesicle transmission has not been investigated systematically. In this study, we performed electrophysiological recordings in calyx of Held synapses. We found that excitatory postsynaptic current (EPSC) and miniature EPSC (mEPSC) amplitudes were unchanged in dysbindin-deficient synapses, but readily releasable pool (RRP) size and calcium dependent vesicle replenishment were affected during high-frequency stimulation. Moreover, dysbindin loss accompanied slightly decreases in Munc18-1 and snapin expression levels, which are associated with vesicle priming and synaptic homeostasis under high-frequency stimulation. Together, we inferred that dysbindin directly interacts with Munc18-1 and snapin to mediate calcium dependent RRP replenishment. Dysbindin loss may lead to RRP replenishment dysregulation during high-frequency stimulation, potentially causing cognitive impairment in schizophrenia.