Clustered dynamics of inhibitory synapses and dendritic spines in the adult neocortex

A key feature of the mammalian brain is its capacity to adapt in response to experience, in part by remodeling of synaptic connections between neurons. Excitatory synapse rearrangements have been monitored in vivo by observation of dendritic spine dynamics, but lack of a vital marker for inhibitory synapses has precluded their observation. Here, we simultaneously monitor in vivo inhibitory synapse and dendritic spine dynamics across the entire dendritic arbor of pyramidal neurons in the adult mammalian cortex using large-volume, high-resolution dual-color two-photon microscopy. We find that inhibitory synapses on dendritic shafts and spines differ in their distribution across the arbor and in their remodeling kinetics during normal and altered sensory experience. Further, we find inhibitory synapse and dendritic spine remodeling to be spatially clustered and that clustering is influenced by sensory input. Our findings provide in vivo evidence for local coordination of inhibitory and excitatory synaptic rearrangements.     

Functional alterations in gut contractility after connexin36 ablation and evidence for gap junctions forming electrical synapses between nitrergic enteric neurons

Neurons in the enteric nervous system utilize numerous neurotransmitters to orchestrate rhythmic gut smooth muscle contractions. We examined whether electrical synapses formed by gap junctions containing connexin36 also contribute to communication between enteric neurons in mouse colon. Spontaneous contractility properties and responses to electrical field stimulation and cholinergic agonist were altered in gut from connexin36 knockout vs. wild-type mice. Immunofluorescence revealed punctate labelling of connexin36 that was localized at appositions between somata of enteric neurons immunopositive for the enzyme nitric oxide synthase. There is indication for a possible functional role of gap junctions between inhibitory nitrergic enteric neurons.     

Impaired electrical signaling disrupts gamma frequency oscillations in connexin 36-deficient mice

Neural processing occurs in parallel in distant cortical areas even for simple perceptual tasks. Associated cognitive binding is believed to occur through the interareal synchronization of rhythmic activity in the gamma (30-80 Hz) range. Such oscillations arise as an emergent property of the neuronal network and require conventional chemical neurotransmission. To test the potential role of gap junction-mediated electrical signaling in this network property, we generated mice lacking connexin 36, the major neuronal connexin. Here we show that the loss of this protein disrupts gamma frequency network oscillations in vitro but leaves high frequency (150 Hz) rhythms, which may involve gap junctions between principal cells (Schmitz et al., 2001), unaffected. Thus, specific connexins differentially deployed throughout cortical networks are likely to regulate different functional aspects of neuronal information processing in the mature brain.     

Persistent activity in neural networks with dynamic synapses

Persistent activity states (attractors), observed in several neocortical areas after the removal of a sensory stimulus, are believed to be the neuronal basis of working memory. One of the possible mechanisms that can underlie persistent activity is recurrent excitation mediated by intracortical synaptic connections. A recent experimental study revealed that connections between pyramidal cells in prefrontal cortex exhibit various degrees of synaptic depression and facilitation. Here we analyze the effect of synaptic dynamics on the emergence and persistence of attractor states in interconnected neural networks. We show that different combinations of synaptic depression and facilitation result in qualitatively different network dynamics with respect to the emergence of the attractor states. This analysis raises the possibility that the framework of attractor neural networks can be extended to represent time-dependent stimuli.     

Dense inhibitory connectivity in neocortex

The connectivity diagram of neocortical circuits is still unknown, and there are conflicting data as to whether cortical neurons are wired specifically or not. To investigate the basic structure of cortical microcircuits, we use a two-photon photostimulation technique that enables the systematic mapping of synaptic connections with single-cell resolution. We map the inhibitory connectivity between upper layers somatostatin-positive GABAergic interneurons and pyramidal cells in mouse frontal cortex. Most, and sometimes all, inhibitory neurons are locally connected to every sampled pyramidal cell. This dense inhibitory connectivity is found at both young and mature developmental ages. Inhibitory innervation of neighboring pyramidal cells is similar, regardless of whether they are connected among themselves or not. We conclude that local inhibitory connectivity is promiscuous, does not form subnetworks, and can approach the theoretical limit of a completely connected synaptic matrix.     

Functional asymmetry of the neocortex electrical activity during food conditioning in dogs

Four dogs were trained to perform a conditioned alimentary response to a sound stimulus. The EEG was recorded from six pairs of chronically implanted neocortical electrodes. The EEG spectra and coherence functions between the neighboring derivations of each of the hemispheres were analyzed in the theta, alpha, beta 1 and beta 2 frequency ranges. At the first stages of conditioning, the percent of cases increased when the highest mean values of EEG frequency were localized in the left hemisphere. Later on the percent of cases, when the mean coherence values in the left hemisphere were higher than in the right hemisphere, also increased. At the stage of conditioned response stabilization, this asymmetry either disappeared or the right hemisphere became more active than the left one. The spatial localization of the maximal values of the EEG frequency was different for different frequency ranges. The highest values in the beta 1 range were more frequently registered in the posterior cortical regions and in the beta 2 range they were revealed, predominantly, in the anterior areas. The maximal values of coherence dominated in the anterior regions and their spatial distribution was similar for different frequencies. Thus, the initial stages of conditioning are accompanied by activation of the left hemisphere.     

Immunohistochemical detection of the neuronal connexin36 in the mouse central nervous system in comparison to connexin36-deficient tissues

Investigating the spatial and temporal expression of connexin36 (Cx36) protein in neuronal tissue is of prime importance to understand the molecular mechanisms underlying extensive electrical coupling. Although Cx36 mRNA was shown to be expressed in neurons of the central nervous system in different studies, only the determination of Cx36 protein expression allows a correlation between localization and its functional role in gap junction-mediated neuronal coupling. After the initial use of antibodies recognizing the skate connexin35 protein, antibodies directed to the mammalian Cx36 sequence allowed the detailed investigation of Cx36 cellular localization. However, results on Cx36 protein distribution still remained controversial in some areas of the central nervous system. In the present study, we have investigated: (a) the distribution of Cx36 protein in various areas of the central nervous system and (b) determined the specificity in the immunohistochemical staining of two polyclonal antibodies comparing wildtype and Cx36-deficient mice. In some areas of the central nervous system, for example in the retina and the inferior nuclear olivary complex, Cx36 antibodies were highly specific, and in the cerebellar cortex, Cx36 protein expression was partly specific. In other regions, particularly in pyramidal cells of the hippocampal formation, non-specific staining was prevalent, indicating that Cx36 antibodies also recognize proteins other than Cx36 in these tissues. The present results argue for a re-evaluation of many documented immunohistochemical protein distribution patterns and require, not only in connexin research, their assessment using null-mutant animals.     

Determination of the numerical density of perforated synapses in rat neocortex

Summary The numerical density and frequency of perforated synapses in the molecular layer of rat parietal cortex have been determined using 4 procedures in an attempt to overcome problems associated with the size and complex three-dimensional shape of perforated synapses. The following procedures were compared: A, single-section analysis; B, adjacent-section analysis; C, semi-serial-section analysis; and D, complete serial-section analysis. All procedures made use of an unbiased counting rule.Estimates of the numerical density of perforated synapses ranged from 0.06 to 0.27×10⁹ mm^-3, and that of all synapses (non-perforated and perforated) from 1.88 to 2.50×10⁹ mm^-3. The frequency of perforated synapses varied from 4.5 to 18.0%. Procedures B (adjacent-section analysis) and D (complete serial-section analysis), neither of which utilize assumptions regarding the shape of synapses, produced comparable results (numerical density of perforated synapses 0.19–0.27×10⁹ mm^-3, and of all synapses 2.24–2.45×10⁹ mm^-3; frequency of perforated synapses 8.6–10.9%). The frequency of perforated synapses appeared to be underestimated by procedure A (single section analysis; 4.5%) and overestimated by C (semi-serial section analysis; 18%).It is concluded that adjacent-section analysis is the most efficient and effective procedure for determining the numerical density and frequency of complex particles, such as perforated synapses. There is, however, no significant difference in the performance of this procedure compared with that of single-section analysis, for determining the numerical density of synapses in general. Nevertheless, inherent problems of bias within the single-section procedure make the adjacent section method the procedure of choice.     

The biochemical anatomy of cortical inhibitory synapses

Classical electron microscopic studies of the mammalian brain revealed two major classes of synapses, distinguished by the presence of a large postsynaptic density (PSD) exclusively at type 1, excitatory synapses. Biochemical studies of the PSD have established the paradigm of the synapse as a complex signal-processing machine that controls synaptic plasticity. We report here the results of a proteomic analysis of type 2, inhibitory synaptic complexes isolated by affinity purification from the cerebral cortex. We show that these synaptic complexes contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of type 1 and type 2 synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain.     

GABAA receptor trafficking-mediated plasticity of inhibitory synapses

Proper developmental, neural cell-type-specific, and activity-dependent regulation of GABAergic transmission is essential for virtually all aspects of CNS function. The number of GABA(A) receptors in the postsynaptic membrane directly controls the efficacy of GABAergic synaptic transmission. Thus, regulated trafficking of GABA(A) receptors is essential for understanding brain function in both health and disease. Here we summarize recent progress in the understanding of mechanisms that allow dynamic adaptation of cell surface expression and postsynaptic accumulation and function of GABA(A) receptors. This includes activity-dependent and cell-type-specific changes in subunit gene expression, assembly of subunits into receptors, as well as exocytosis, endocytic recycling, diffusion dynamics, and degradation of GABA(A) receptors. In particular, we focus on the roles of receptor-interacting proteins, scaffold proteins, synaptic adhesion proteins, and enzymes that regulate the trafficking and function of receptors and associated proteins. In addition, we review neuropeptide signaling pathways that affect neural excitability through changes in GABA(A)R trafficking.     

Co-existent activity patterns in inhibitory neuronal networks with short-term synaptic depression

A network of two neurons mutually coupled through inhibitory synapses that display short-term synaptic depression is considered. We show that synaptic depression expands the number of possible activity patterns that the network can display and allows for co-existence of different patterns. Specifically, the network supports different types of n-m anti-phase firing patterns, where one neuron fires n spikes followed by the other neuron firing m spikes. When maximal synaptic conductances are identical, n-n anti-phase firing patterns are obtained and there are conductance intervals over which different pairs of these solutions co-exist. The multitude of n-m anti-phase patterns and their co-existence are not found when the synapses are non-depressing. Geometric singular perturbation methods for dynamical systems are applied to the original eight-dimensional model system to derive a set of one-dimensional conditions for the existence and co-existence of different anti-phase solutions. The generality and validity of these conditions are demonstrated through numerical simulations utilizing the Hodgkin-Huxley and Morris-Lecar neuronal models.     

Axo-glial synapses and GABA-accumulating glial cells in the embryonic neocortex of the rat

Summary On embryonic day 18, synapse-like contacts are found on certain non-neuronal cells appearing in clusters in lamina I (LI) of the parieto-occipital cortex of the rat. The structural criteria of these cells resemble those of immature glial cells: (1) The elongated nuclei containing dispersed chromatin are enclosed by a membrane showing narrow folds. (2) The cytoplasm contains many free ribosomes and a few dilated cisterns of the rough endoplasmic reticulum with granular or filamentous contents. (3) The plasma membrane forms concave adaptations toward neighboring neuronal processes. (4) At least one of the processes makes contact with the basal lamina of a vessel wall. The presynaptic elements contain a varying number of synaptic vesicles, and the pre- and postsynaptic membranes show densifications. Certain neurons and glial cells of the neocortex have the capability to accumulate GABA at day 16 of embryonic life. Only the more differentiated glial cells accumulate GABA. Many of these elements closely resemble the glial cells receiving synapse-like contacts, e.g., with respect to their cytological characteristics, clustering, and laminar position. According to recent experiments with adult ganglion cells, GABA released from glial cells might promote synaptogenesis by increasing the number of postsynaptic thickenings on the surrounding neurons. Thus, it cannot be excluded that transitory axo-glial synapses, by inducing GABA release, play a specific role in the earliest stages of synaptogenesis.     

The antihypertensive activity of angiotensin-converting enzyme inhibitory peptide containing in bovine lactoferrin

Angiotensin I-converting enzyme (ACE) inhibitory peptide was isolated from the bovine lactoferrin hydrolysate using peptic hydrolysis by 2-step of reverse-phase high-performance liquid chromatography. This peptide was identified as Leu-Arg-Pro-Val-Ala-Ala and it produced a concentration-dependent inhibition of ACE activity in vitro with an IC50 value of about 4.14 microM. Also, this inhibition was identified as noncompetitive from the Lineweaver-Burk plot. Moreover, the antihypertensive activity of Leu-Arg-Pro-Val-Ala-Ala was investigated by the intravenous injection into spontaneously hypertensive rats (SHRs). A dose-dependent reduction of systolic blood pressure by this peptide was observed at 60 min after injection and it maximally decreased the blood pressure at a rate of 1 nmol/ml/kg. The blood pressure lowering activity of this peptide was calculated as 210% of captopril (10 pmol/ml/kg) that was used as positive control. Otherwise, identification of this peptide in the blood of SHRs was carried out chromatographically. Reduction of blood pressure coincides with the peak peptide concentration in the serum. Thus, we conclude that this peptide inhibits ACE activity in vitro and lowers systolic blood pressure in spontaneously hypertensive rat.     

Differences in the cytoskeleton in inhibitory and excitatory synapses

The cytoskeleton of afferent chemical synapses, with various ultrastructure of contact zones, was examined in the Mauthner cells of the goldfish. The synapses with combined active zones and desmosome-like specialized contacts possessed a well developed cytoskeleton consisting of filaments and microtubules oriented towards the synaptic apposition. Regular arrays of synaptic vesicles oriented in the same direction were observed beyond and near the active zones. The cytoskeleton of the synapses lacking desmosome-like formations was diffusely organized throughout the boutons. The distribution of vesicles in the vicinity of active zones was also not ordered. The role of cytoskeleton in organization of the two morphologically distinct synapses is discussed. A special function of cytoskeleton as an intermediary between synaptoplasm and membrane is regarded as a necessary basis for plasticity of excitatory rather than inhibitory synapses.     

Gain adjustment of inhibitory synapses in the auditory system

A group of central auditory neurons residing in the lateral superior olivary nucleus (LSO) responds selectively to interaural level differences and may contribute to sound localization. In this simple circuit, ipsilateral sound increases firing of LSO neurons, whereas contralateral sound inhibits the firing rate via activation of the medial nucleus of the trapezoid body (MNTB). During development, individual MNTB fibers arborize within the LSO, but they undergo a restriction of their boutons that ultimately leads to mature topography. A critical issue is whether a distinct form of inhibitory synaptic plasticity contributes to MNTB synapse elimination within LSO. Whole-cell recording from LSO neurons in brain slices from developing gerbils show robust long-term depression (LTD) of the MNTB-evoked IPSP/Cs when the MNTB was activated at a low frequency (1 Hz). These inhibitory synapses also display mixed GABA/glycinergic transmission during development, as assessed physiologically and immunohistochemically (Kotak et al. 1998). While either glycine or GABA_A receptors could independently display inhibitory LTD, focal delivery of GABA, but not glycine, at the postsynaptic-locus induces depression. Furthermore, the GABA_B receptor antagonist, SCH-50911, prevents GABA or synaptically induced depression. Preliminary evidence also indicated strengthening of inhibitory transmission (LTP) by a distinct pattern of inhibitory activity. These data support the idea that GABA is crucial for the expression inhibitory LTD and that this plasticity may underlie the early refinement of inhibitory synaptic connections in the LSO.     

Presynaptic NMDA receptors mediate IPSC potentiation at GABAergic synapses in developing rat neocortex

Background

NMDA receptors are traditionally viewed as being located postsynaptically, at both synaptic and extrasynaptic locations. However, both anatomical and physiological studies have indicated the presence of NMDA receptors located presynaptically. Physiological studies of presynaptic NMDA receptors on neocortical GABAergic terminals and their possible role in synaptic plasticity are lacking.

Methodology/Principal Findings

We report here that presynaptic NMDA receptors are present on GABAergic terminals in developing (postnatal day (PND) 12-15) but not older (PND21-25) rat frontal cortex. Using MK-801 in the recording pipette to block postsynaptic NMDA receptors, evoked and miniature IPSCs were recorded in layer II/III pyramidal cells in the presence of AMPA/KA receptor antagonists. Bath application of NMDA or NMDA receptor antagonists produced increases and decreases in mIPSC frequency, respectively. Physiologically patterned stimulation (10 bursts of 10 stimuli at 25 Hz delivered at 1.25 Hz) induced potentiation at inhibitory synapses in PND12-15 animals. This consisted of an initial rapid, large increase in IPSC amplitude followed by a significant but smaller persistent increase. Similar changes were not observed in PND21-25 animals. When 20 mM BAPTA was included in the recording pipette, potentiation was still observed in the PND12-15 group indicating that postsynaptic increases in calcium were not required. Potentiation was not observed when patterned stimulation was given in the presence of D-APV or the NR2B subunit antagonist Ro25-6981.

Conclusions/Significance

The present results indicate that presynaptic NMDA receptors modulate GABA release onto neocortical pyramidal cells. Presynaptic NR2B subunit containing NMDA receptors are also involved in potentiation at developing GABAergic synapses in rat frontal cortex. Modulation of inhibitory GABAergic synapses by presynaptic NMDA receptors may be important for proper functioning of local cortical networks during development.