Vesicular glutamate transporter type 3 (VGLUT3) containing neuronal elements were characterized using antibodies to VGLUT3 and molecular cell markers. All VGLUT3‐positive somata were immunoreactive ...for CCK, and very rarely, also for calbindin; none was positive for parvalbumin, calretinin, VIP or somatostatin. In the CA1 area, 26.8 ± 0.7% of CCK‐positive interneuron somata were VGLUT3‐positive, a nonoverlapping 22.8 ± 1.9% were calbindin‐positive, 10.7 ± 2.5% VIP‐positive and the rest were only CCK‐positive. The patterns of coexpression were similar in the CA3 area, the dentate gyrus and the isocortex. Immunoreactivity for VGLUT3 was undetectable in pyramidal and dentate granule cells. Boutons colabelled for VGLUT3, CCK and GAD were most abundant in the cellular layers of the hippocampus and in layers II–III of the isocortex. Large VGLUT3‐labelled boutons at the border of strata radiatum and lacunosum‐moleculare in the CA1 area were negative for GAD, but were labelled for vesicular monoamine transporter type 2, plasmalemmal serotonin transporter or serotonin. No colocalization was found in terminals between VGLUT3 and parvalbumin, vesicular acetylcholine transporter and group III (mGluR7a,b; mGluR8a,b) metabotropic glutamate receptors. In stratum radiatum and the isocortex, VGLUT3‐positive but GAD‐negative boutons heavily innervated the soma and proximal dendrites of some VGLUT3‐ or calbindin‐positive interneurons. The results suggest that boutons coexpressing VGLUT3, CCK and GAD originate from CCK‐positive basket cells, which are VIP‐immunonegative. Other VGLUT3‐positive boutons immunopositive for serotonergic markers but negative for GAD probably originate from the median raphe nucleus and innervate select interneurons. The presumed amino acid substrate of VGLUT3 may act on presynaptic kainate or group II metabotropic glutamate receptors.
Group III metabotropic glutamate receptors (mGluRs) are selectively activated by
L-2-amino-4-phosphonobutyrate (
L-AP4), which produces depression of synaptic transmission. The relative contribution ...of different group III mGluRs to the effects of
L-AP4 remains to be clarified. Here, we assessed the distribution of mGluR4 in the rat and mouse brain using affinity-purified antibodies raised against its entire C-terminal domain. The antibodies reacted specifically with mGluR4 and not with other mGluRs in transfected COS 7 cells. No immunoreactivity was detected in brains of mice with gene-targeted deletion of mGluR4. Pre-embedding immunocytochemistry for light and electron microscopy showed the most intense labelling in the cerebellar cortex, basal ganglia, the sensory relay nuclei of the thalamus, and some hippocampal areas. Immunolabelling was most intense in presynaptic active zones. In the basal ganglia, both the direct and indirect striatal output pathways showed immunolabelled terminals forming mostly type II synapses on dendritic shafts. The localisation of mGluR4 on GABAergic terminals of striatal projection neurones suggests a role as a presynaptic heteroreceptor. In the cerebellar cortex and hippocampus, mGluR4 was also localised in terminals establishing type I synapses, where it probably operates as an autoreceptor. In the hippocampus, mGluR4 labelling was prominent in the dentate molecular layer and CA1–3 strata lacunosum moleculare and oriens. Somatodendritic profiles of some stratum oriens/alveus interneurones were richly decorated with mGluR4-labelled axon terminals making either type I or II synapses. This differential localisation suggests a regulation of synaptic transmission via a target cell-dependent synaptic segregation of mGluR4.
Our results demonstrate that, like other group III mGluRs, presynaptic mGluR4 is highly enriched in the active zone of boutons innervating specific classes of neurones. In addition, the question of alternatively spliced mGluR4 isoforms is discussed.
In neocortical circuits, repetitively active neurons evoke unitary postsynaptic potentials (PSPs) whose peak amplitudes either increase (facilitate) or decrease (depress) progressively. To examine ...the basis for these different synaptic responses, we made simultaneous recordings from three classes of neurons in cortical layer 2/3. We induced repetitive action potentials in pyramidal cells and recorded the evoked unitary excitatory (E)PSPs in two classes of GABAergic neurons. We observed facilitation of EPSPs in bitufted GABAergic interneurons, many of which expressed somatostatin immunoreactivity. EPSPs recorded from multipolar interneurons, however, showed depression. Some of these neurons were immunopositive for parvalbumin. Unitary inhibitory (I)PSPs evoked by repetitive stimulation of a bitufted neuron also showed a less pronounced but significant difference between the two target neurons. Facilitation and depression involve presynaptic mechanisms, and because a single neuron can express both behaviors simultaneously, we infer that local differences in the molecular structure of presynaptic nerve terminals are induced by retrograde signals from different classes of target neurons. Because bitufted and multipolar neurons both formed reciprocal inhibitory connections with pyramidal cells, the results imply that the balance of activation between two recurrent inhibitory pathways in the neocortex depends on the frequency of action potentials in pyramidal cells.
Glutamate is a major neurotransmitter in the brain that acts both through fast ionotropic receptors and through slower metabotropic receptors coupled to G proteins. Both receptors are present ...throughout the somatodendritic domain of neurons as shown by immunohistochemical and patch clamp recording studies. Immunogold labelling revealed a concentration of metabotropic receptors at the edge, but not within the main body of anatomically defined synapses, raising the possibility that ionotropic and metabotropic receptors are segregated. We applied double immunogold labelling to study glutamatergic parallel and climbing fibre synapses in the cerebellar cortex. The ionotropic AMPA type receptors occupy the membrane opposite the release site in the main body of the synaptic junction, whereas the metabotropic receptors are located at the periphery of the same synapses. Furthermore, immunoreactivity for AMPA receptors is at least twice as high in the parallel fibre synapses as in glutamatergic mossy fibre synapses. We suggest that the spatial segregation of ionotropic and metabotropic glutamate receptors permits the differential activation of these receptors according to the amount of glutamate released presynaptically, whereas the different densities of the ionotropic receptor at distinct synapses could allow the same amount of glutamate to evoke fast responses of different magnitude.
The cerebral cortex encodes, stores and combines information about the internal and external environment in rhythmic activity
of multiple frequency ranges. Neurones of the cortex can be defined, ...recognized and compared on the comprehensive application
of the following measures: (i) brain area- and cell domain-specific distribution of input and output synapses, (ii) expression
of molecules involved in cell signalling, (iii) membrane and synaptic properties reflecting the expression of membrane proteins,
(iv) temporal structure of firing in vivo , resulting from (i)â(iii). Spatial and temporal measures of neurones in the network reflect an indivisible unity of evolutionary
design, i.e. neurones do not have separate structure or function. The blueprint of this design is most easily accessible in
the CA1 area of the hippocampus, where a relatively uniform population of pyramidal cells and their inputs follow an instantly
recognizable laminated pattern and act within stereotyped network activity patterns. Reviewing the cell types and their spatio-temporal
interactions, we suggest that CA1 pyramidal cells are supported by at least 16 distinct types of GABAergic neurone. During
a given behaviour-contingent network oscillation, interneurones of a given type exhibit similar firing patterns. During different network oscillations representing two distinct brain states, interneurones of the same
class show different firing patterns modulating their postsynaptic target-domain in a brain-state-dependent manner. These results suggest roles
for specific interneurone types in structuring the activity of pyramidal cells via their respective target domains, and accurately
timing and synchronizing pyramidal cell discharge, rather than providing generalized inhibition. Finally, interneurones belonging
to different classes may fire preferentially at distinct time points during a given oscillation. As different interneurones
innervate distinct domains of the pyramidal cells, the different compartments will receive GABAergic input differentiated
in time. Such a dynamic, spatio-temporal, GABAergic control, which evolves distinct patterns during different brain states,
is ideally suited to regulating the input integration of individual pyramidal cells contributing to the formation of cell
assemblies and representations in the hippocampus and, probably, throughout the cerebral cortex.
Two types of GABAergic interneurone are known to express cholecystokinin-related peptides in the isocortex: basket cells, which preferentially innervate the somata and proximal dendrites of pyramidal ...cells; and double bouquet cells, which innervate distal dendrites and dendritic spines. In the hippocampus, cholecystokinin immunoreactivity has only been reported in basket cells. However, at least eight distinct GABAergic interneurone types terminate in the dendritic domain of CA1 pyramidal cells, some of them with as yet undetermined neurochemical characteristics. In order to establish whether more than one population of cholecystokinin-expressing interneurone exist in the hippocampus, we have performed whole-cell current clamp recordings from interneurones located in the stratum radiatum of the hippocampal CA1 region of developing rats. Recorded neurones were filled with biocytin to reveal their axonal targets, and were tested for the presence of pro-cholecystokinin immunoreactivity.
The results show that two populations of cholecystokinin-immunoreactive interneurones exist in the CA1 area (
n=15 positive cells). Cholecystokinin-positive basket cells (53%) preferentially innervate stratum pyramidale and adjacent strata oriens and radiatum. A second population of cholecystokinin-positive cells, previously described as Schaffer collateral-associated interneurones Vida et al. (1998) J. Physiol. 506, 755–773, have axons that ramify almost exclusively in strata radiatum and oriens, overlapping with the Schaffer collateral/commissural pathway originating from CA3 pyramidal cells. Two of seven of the Schaffer collateral-associated cells were also immunopositive for calbindin. Soma position and orientation in stratum radiatum, the number and orientation of dendrites, and the passive and active membrane properties of the two cell populations are only slightly different. In addition, in stratum radiatum and its border with lacunosum of perfusion-fixed hippocampi, 31.6±3.8% (adult) or 26.8±2.9% (postnatal day 17–20) of cholecystokinin-positive cells were also immunoreactive for calbindin.
Therefore, at least two populations of pro-cholecystokinin-immunopositive interneurones, basket and Schaffer collateral-associated cells, exist in the CA1 area of the hippocampus, and are probably homologous to cholecystokinin-immunopositive basket and double bouquet cells in the isocortex. It is not known if the GABAergic terminals of double bouquet cells are co-aligned with specific glutamatergic inputs. However, in the hippocampal CA1 area, it is clear that the terminals of Schaffer collateral-associated cells are co-stratified with the glutamatergic input from the CA3 area, with as yet unknown functional consequences. The division of the postsynaptic neuronal surface by two classes of GABAergic cell expressing cholecystokinin in both the hippocampus and isocortex provides further evidence for the uniform synaptic organisation of the cerebral cortex.
1. The effects of synapses established by smooth dendritic neurones on pyramidal and spiny stellate cells were studied in
areas 17 and 18 of the cat visual cortex in vitro. Paired intracellular ...recordings with biocytin-filled electrodes and subsequent
light and electron microscopic analysis were used to determine the sites of synaptic interaction. 2. All smooth dendritic
cells established type II synapses previously shown to be made by terminals containing GABA, therefore the studied cells are
probably GABAergic. Three classes of presynaptic cell could be defined, based on their efferent synaptic target preference
determined from random samples of unlabelled postsynaptic cells. (a) Basket cells (n = 6) innervated mainly somata (49.9 +/-
13.8%) and dendritic shafts (45.2 +/- 10.7%) and, to a lesser extent, dendritic spines (4.9 +/- 4.6%). (b) Dendrite-targeting
cells (n = 5) established synapses predominantly on dendritic shafts (84.3 +/- 9.4%) and less frequently on dendritic spines
(11.2 +/- 6.7%) or somata (4.5 +/- 4.7%). (c) Double bouquet cells (n = 4) preferred dendritic spines (69.2 +/- 4.2%) to dendritic
shafts (30.8 +/- 4.2%) as postsynaptic targets and avoided somata. 3. Interneurones formed 5240 +/- 1600 (range, 2830-9690)
synaptic junctions in the slices. Based on the density of synapses made by single interneurones and the volume density of
GABAergic synapses, it was calculated that an average interneurone provides 0.66 +/- 0.20% of the GABAergic synapses in its
axonal field. 4. The location of synaptic junctions on individual, identified postsynaptic cells reflected the overall postsynaptic
target distribution of the same GABAergic neurone. The number of synaptic junctions between pairs of neurones could not be
predicted from light microscopic examination. The number of electron microscopically verified synaptic sites was generally
smaller for the dendritic domain and larger for the somatic domain than expected from light microscopy. All presynaptic cells
established multiple synaptic junctions on their postsynaptic target cells. A basket cell innervated a pyramidal cell via
fifteen release sites; the numbers of synapses formed by three dendrite-targeting cells on pyramidal cells were seventeen
and eight respectively, and three on a spiny stellate cell; the interaction between a double bouquet cell and a postsynaptic
pyramidal cell was mediated by ten synaptic junctions. 5. All three types of interneurone (n = 6; 2 for each type of cell)
elicited short-latency IPSPs with fast rise time (10-90%; 2.59 +/- 1.02 ms) and short duration (at half-amplitude, 15.82 +/-
5.24 ms), similar to those mediated by GABAA receptors. 6. Average amplitudes of unitary IPSPs (n = 6) were 845 +/- 796 microV
(range, 134-2265 microV). Variability of IPSP amplitude was moderate, the average ratio of IPSP and baseline noise variance
was 1.54 +/- 0.96. High frequency activation of single presynaptic dendrite-targeting cells led to an initial summation followed
by use-dependent depression of the averaged postsynaptic response. Double bouquet cell-evoked IPSPs, recorded in the soma,
had a smaller amplitude than those evoked by the other two cell types. In all connections, transmission failures were rare
or absent, particularly when mediated by a high number of release sites. 7. The results demonstrate that different types of
neocortical GABAergic neurones innervate distinct domains on the surface of their postsynaptic target cells. Nevertheless,
all three types of cell studied here elicit fast IPSPs and provide GABAergic input through multiple synaptic release sites
with few, if any, failures of transmission.
Five acylation reagents have been compared for use as derivatizing agents for the analysis of amphetamine-type stimulants (ATS) in urine by gas chromatography-mass spectrometry (GC-MS). The evaluated ...reagents were heptafluorobutyric anhydride, pentafluoropropionic anhydride, trifluoroacetic anhydride, acetic anhydride (AA) and N-methyl-bis(trifluoroacetamide). The ATS included amphetamine, methamphetamine (MA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA). A mixture of the ATS was added to urine (1 mL) followed by KOH solution and saturated NaHCO3 solution. The sample was then extracted with dichloromethane and the derivatizing agent and 2 µL were injected into the GC-MS instrument. The derivatizing agents were compared with reference to the signal-to-noise (S/N) ratios, peak area values, relative standard deviations (RSDs), linearities, limits of detection (LODs) and selectivities. The acetic anhydride proved to be the best according to the S/N ratio and peak area results for amphetamine, MA, MDMA and MDEA. The best RSD values of peak areas and of S/N ratios at 3 µg/mL were also given by AA in cases of MDA, MDMA and MDEA. At 20 µg/mL, the lowest RSD values of peak areas for MDA and the lowest RSD values of S/N ratios for MA, MDA, MDMA and MDEA were again given by AA. Additionally, the highest correlation coefficients for MA, MDA, MDMA and MDEA and the lowest LOD results for MA, MDMA and MDEA were produced by AA.
SYNCHRONIZATION of neuronal activity is fundamental in the operation of cortical networks. With respect to an ongoing synchronized oscillation, the precise timing of action potentials is an ...attractive candidate mechanism for information coding. Networks of inhibitory interneurons have been proposed to have a role in entraining cortical, synchronized 40-Hz activity. Here we demonstrate that individual GABAergic interneurons can effectively phase spontaneous firing and subthreshold oscillations in hippocampal pyramidal cells at 0 frequencies (4-7 Hz). The efficiency of this entrainment is due to interaction of GABAA-receptor-mediated hyperpolarizing synaptic events with intrinsic oscillatory mechanisms tuned to this frequency range in pyramidal cells. Moreover, this GABAergic mechanism is sufficient to synchronize the firing of pyramidal cells. Thus, owing to the divergence of each GABAergic interneuron, more than a thousand pyramidal cells may share a common temporal reference established by an individual interneuron.
Understanding any brain circuit will require a categorization of its constituent neurons. In hippocampal area CA1, at least 23 classes of GABAergic neuron have been proposed to date. However, this ...list may be incomplete; additionally, it is unclear whether discrete classes are sufficient to describe the diversity of cortical inhibitory neurons or whether continuous modes of variability are also required. We studied the transcriptomes of 3,663 CA1 inhibitory cells, revealing 10 major GABAergic groups that divided into 49 fine-scale clusters. All previously described and several novel cell classes were identified, with three previously described classes unexpectedly found to be identical. A division into discrete classes, however, was not sufficient to describe the diversity of these cells, as continuous variation also occurred between and within classes. Latent factor analysis revealed that a single continuous variable could predict the expression levels of several genes, which correlated similarly with it across multiple cell types. Analysis of the genes correlating with this variable suggested it reflects a range from metabolically highly active faster-spiking cells that proximally target pyramidal cells to slower-spiking cells targeting distal dendrites or interneurons. These results elucidate the complexity of inhibitory neurons in one of the simplest cortical structures and show that characterizing these cells requires continuous modes of variation as well as discrete cell classes.
PDF
