Postsynaptic targets of somatostatin-containing interneurons in the rat basolateral amygdala

The basolateral amygdala contains several subpopulations of inhibitory interneurons that can be distinguished on the basis of their content of calcium-binding proteins or peptides. Although previous studies have shown that interneuronal subpopulations containing parvalbumin (PV) or vasoactive intestinal peptide (VIP) innervate distinct postsynaptic domains of pyramidal cells as well as other interneurons, very little is known about the synaptic outputs of the interneuronal subpopulation that expresses somatostatin (SOM). The present study utilized dual-labeling immunocytochemical techniques at the light and electron microscopic levels to analyze the innervation of pyramidal cells, PV+ interneurons, and VIP+ interneurons in the anterior basolateral amygdalar nucleus (BLa) by SOM+ axon terminals. Pyramidal cell somata and dendrites were selectively labeled with antibodies to calcium/calmodulin-dependent protein kinase II (CaMK); previous studies have shown that the vast majority of dendritic spines, whether CAMK+ or not, arise from pyramidal cells. Almost all SOM+ axon terminals formed symmetrical synapses. The main postsynaptic targets of SOM+ terminals were small-caliber CaMK+ dendrites and dendritic spines, some of which were CaMK+. These SOM+ synapses with dendrites were often in close proximity to asymmetrical (excitatory) synapses to these same structures formed by unlabeled terminals. Few SOM+ terminals formed synapses with CaMK+ pyramidal cell somata or large-caliber (proximal) dendrites. Likewise, only 15% of SOM+ terminals formed synapses with PV+, VIP+, or SOM+ interneurons. These findings suggest that inhibitory inputs from SOM+ interneurons may interact with excitatory inputs to pyramidal cell distal dendrites in the BLa. These interactions might affect synaptic plasticity related to emotional learning.     

3D electron microscopic reconstruction of segments of rat cerebellar purkinje cell dendrites receiving ascending and parallel fiber granule cell synaptic inputs

Growing physiological evidence suggests that there are functional differences between synapses made by the ascending and parallel fiber segments of the granule axon on cerebellar Purkinje cells. Supporting this view, our previous electron microscopic studies suggested that these synapses also contacted different regions of the Purkinje cell dendrite, and in particular that ascending segment synapses are made exclusively on the smallest diameter Purkinje cell dendrites. In the current study we used serial electron microscopic techniques to reconstruct Purkinje cell dendritic segments up to almost 10 μm in length. Using a combination of anatomical and immunological labeling techniques we identified the ascending or parallel fiber origins of the excitatory synaptic inputs onto dendritic spines, as well as the location of inhibitory synapses made directly on the dendritic shaft. The results confirmed that there are regions of the Purkinje cell dendrite receiving exclusively ascending or parallel fiber synapses and that ascending segment synapses are only found on small‐diameter dendrites. In addition, we describe for the first time small‐diameter dendritic regions contacted by both types of excitatory synapses. While our data suggest that the majority of inhibitory inputs to the Purkinje cell tree are associated with parallel fiber synaptic inputs, we also found inhibitory inputs on dendritic regions with mixed ascending and parallel fiber inputs, or exclusively parallel fiber inputs. The finding that ascending and parallel fiber inputs can be segregated on the Purkinje cell dendritic tree provides further evidence that these excitatory granule cell synaptic inputs may be functionally distinct. J. Comp. Neurol. 514:583–594, 2009. © 2009 Wiley‐Liss, Inc.     

Ultrastructural study of cholecystokinin-immunoreactive cells and processes in area CA1 of the rat hippocampus

We used light and electron microscopic immunocytochemical methods to examine the structure of neuronal perikarya and processes containing cholecystokinin-like immunoreactivity (CCK-IR) in area CA1 of the rat hippocampus. The morphology of stained perikarya, their positions within all laminae, and the orientation of their dendrites indicate that CCK-IR is located in interneurons. These cells were seen in the electron microscope to have deeply folded nuclei and to receive both symmetric and asymmetric synaptic junctions on their cell somata and dendritic shafts. Their dendrites are essentially spine-free, but form bulges at the site of some asymmetric synaptic junctions. Axonal varicosities containing CCK-IR make symmetric synaptic junctions with cell somata and dendritic shafts of both pyramidal and non-pyramidal neurons. In addition, CCK-IR varicosities form symmetric junctions with unstained non-pyramidal neurons and with CCK-IR cells, suggesting either recurrent innervation of one cell on itself or interaction between interneurons. The presence of CCK-IR varicosities and synaptic junctions on pyramidal cells is in agreement with physiological data which indicate that CCK has a direct postsynaptic action. The observation of CCK-IR varicosities forming synaptic junctions on non-pyramidal cells suggests that CCK might also modify the response of interneurons.     

Map of the synapses onto layer 4 basket cells of the primary visual cortex of the cat

The pattern of excitatory and inhibitory inputs to the inhibitory neurons is largely unknown. We have set out to quantify the major excitatory and inhibitory inputs to layer 4 basket cells from the primary visual cortex of the cat. The synapses formed with the soma, and proximal and distal dendrites, were examined at the light and electron microscopic levels in four basket cells, recorded in vivo and filled with horseradish peroxidase. The major afferents of layer 4 have been well characterised, both at the light and electron microscopic levels. The sizes of the synaptic boutons of the major excitatory inputs to layer 4 from the thalamic relay cells, spiny stellate cells, and layer 6 pyramidal neurons are statistically different. Their distributions were compared to those of the boutons forming asymmetric contacts onto the basket cells, which were assumed to be provided by the same set of excitatory afferents. The best-fit results showed that about equal numbers of synapses were provided by the layer 6 pyramids (43%) and the spiny stellates (44%), whereas the thalamic afferents contributed only 13%. A similar analysis on the symmetric synaptic input to the basket cells indicated that as much as 79% of the symmetric synapses could have originated from other layer 4 basket cells. Thalamic and spiny stellate synapses were preferentially located on the soma and proximal dendrites, regions that also had 76% of all the symmetric contacts. J. Comp. Neurol. 380: 230–242, 1997. © 1997 Wiley-Liss, Inc.     

Synaptic input to dentate granule cell basal dendrites in a rat model of temporal lobe epilepsy

In patients with temporal lobe epilepsy some dentate granule cells develop basal dendrites. The extent of excitatory synaptic input to basal dendrites is unclear, nor is it known whether basal dendrites receive inhibitory synapses. We used biocytin to intracellularly label individual granule cells with basal dendrites in epileptic pilocarpine-treated rats. An average basal dendrite had 3.9 branches, was 612 microm long, and accounted for 16% of a cell's total dendritic length. In vivo intracellular labeling and postembedding GABA-immunocytochemistry were used to evaluate synapses with basal dendrites reconstructed from serial electron micrographs. An average of 7% of 1,802 putative synapses were formed by GABA-positive axon terminals, indicating synaptogenesis by interneurons. Ninety-three percent of the identified synapses were GABA-negative. Most GABA-negative synapses were with spines, but at least 10% were with dendritic shafts. Multiplying basal dendrite length/cell and synapse density yielded an estimate of 180 inhibitory and 2,140 excitatory synapses per granule cell basal dendrite. Based on previous estimates of synaptic input to granule cells in control rats, these findings suggest an average basal dendrite receives approximately 14% of the total inhibitory and 19% of excitatory synapses of a cell. These findings reveal that basal dendrites are a novel source of inhibitory input, but they primarily receive excitatory synapses.     

Pyramidal cells of the rat basolateral amygdala: synaptology and innervation by parvalbumin-immunoreactive interneurons

The generation of emotional responses by the basolateral amygdala is determined largely by the balance of excitatory and inhibitory inputs to its principal neurons, the pyramidal cells. The activity of these neurons is tightly controlled by gamma-aminobutyric acid (GABA)-ergic interneurons, especially a parvalbumin-positive (PV(+)) subpopulation that constitutes almost half of all interneurons in the basolateral amygdala. In the present semiquantitative investigation, we studied the incidence of synaptic inputs of PV(+) axon terminals onto pyramidal neurons in the rat basolateral nucleus (BLa). Pyramidal cells were identified by using calcium/calmodulin-dependent protein kinase II (CaMK) immunoreactivity as a marker. To appreciate the relative abundance of PV(+) inputs compared with excitatory inputs and other non-PV(+) inhibitory inputs, we also analyzed the proportions of asymmetrical (presumed excitatory) synapses and symmetrical (presumed inhibitory) synapses formed by unlabeled axon terminals targeting pyramidal neurons. The results indicate that the perisomatic region of pyramidal cells is innervated almost entirely by symmetrical synapses, whereas the density of asymmetrical synapses increases as one proceeds from thicker proximal dendritic shafts to thinner distal dendritic shafts. The great majority of synapses with dendritic spines are asymmetrical. PV(+) axon terminals form mainly symmetrical synapses. These PV(+) synapses constitute slightly more than half of the symmetrical synapses formed with each postsynaptic compartment of BLa pyramidal cells. These data indicate that the synaptology of basolateral amygdalar pyramidal cells is remarkably similar to that of cortical pyramidal cells and that PV(+) interneurons provide a robust inhibition of both the perisomatic and the distal dendritic domains of these principal neurons.     

Local circuitry involving parvalbumin-positive basket cells in the CA2 region of the hippocampus

There is a growing recognition that the CA2 region of the hippocampus has its own distinctive properties, inputs, and pathologies. The dendritic and axonal patterns of some interneurons in this region are also strikingly different from those described previously in CA1 and CA3. The local circuitry in this region, however, had yet to be studied in detail. Accordingly, using dual intracellular recordings and biocytin-filling, excitatory and inhibitory connections involving CA2 parvalbumin-positive basket cells were characterized for the first time. CA2 basket cells targeted neighboring pyramidal cells and received excitatory inputs from them. CA2 basket cells that resembled those in CA1 with a fast spiking behavior and dendritic tree confined to the region of origin received depressing excitatory postsynaptic potentials (EPSPs). In contrast, unlike CA1 basket cells but like CA1 Oriens-Lacunosum Moleculare (OLM) cells, the majority of CA2 basket cells had horizontally oriented dendrites in Stratum Oriens (SO), which extended into all three CA subfields, had an adapting firing pattern, presented a "sag" in their voltage responses to hyperpolarizing current injection, and received facilitating EPSPs. The expression of I(h) did not influence the EPSP time courses and paired pulse ratios (PPR). Estimates of the probability of release (p) for the depressing and facilitating EPSPs were correlated with the PPR. Connections with low probabilities of release had higher PPR. Quantal amplitude (q) for the facilitating connections was larger than q at depressing inputs onto fast spiking basket cells.     

Subdivisions in the Multiple GABAergic Innervation of Granule Cells in the Dentate Gyrus of the Rat Hippocampus

The sources of GABAergic innervation to granule cells were studied to establish how the basic cortical circuit is implemented in the dentate gyrus. Five types of neuron having extensive local axons were recorded electrophysiologically in vitro and filled intracellularly with biocytin (Han et al., 1993). They were processed for electron microscopy in order to reveal their synaptic organization and postsynaptic targets, and to test whether their terminals contained GABA. (1) The hilar cell, with axon terminals in the commissural and association pathway termination field (HICAP cell), formed Gray's type 2 (symmetrical) synapses with large proximal dendritic shafts (n= 18), two-thirds of which could be shown to emit spines, and with small dendritic branches (n= 6). Other boutons of the HICAP neuron were found to make either Gray's type 1 (asymmetrical) synapses (n= 4) or type 2 synapses (n= 6) with dendritic spines. Using a highly sensitive silver-intensified immunogold method for the postembedding visualization of GABA immunoreactivity, both the terminals and the dendrites of the HICAP cell were found to be immunopositive, whereas its postsynaptic targets were GABA-immunonegative. The dendritic shafts of the HICAP cell received synapses from both GABA-negative and GABA-positive boutons; the dendritic spines which densely covered the main apical dendrite in the medial one-third of the molecular layer received synapses from GABA-negative boutons. (2) The hilar cell, with axon terminals distributed in conjunction with the perforant path termination field (HIPP cell), established type 2 synapses with distal dendritic shafts (n= 17), most of which could be shown to emit spines, small-calibre dendritic profiles (n= 2) and dendritic spines (n= 6), all showing characteristics of granule cell dendrites. The sparsely spiny dendrites of the HIPP cell were covered with many synaptic boutons on both their shafts and their spines. (3) The cell with soma in the molecular layer had an axon associated with the perforant path termination field (MOPP cell). This GABA-immunoreactive cell made type 2 synapses exclusively on dendritic shafts (n= 20), 60% of which could be shown to emit spines. The smooth dendrites of the MOPP cell were also restricted to the outer two-thirds of the molecular layer, where they received both GABA-negative and GABA-positive synaptic inputs. (4) The extensive axonal arborization of the dentate basket cell terminated mainly on somata (n= 26) and proximal dendrites (n= 9) in the granule cell layer, and some boutons made synapses on somatic spines (n= 6); all boutons established type 2 synapses. (5) The dentate axo-axonic cell established type 2 synapses (n= 14) exclusively on axon initial segments of granule cells in the granule cell layer, and on initial segments of presumed mossy cells in the hilus. The results demonstrate that granule cells receive inputs from the local circuit axons of at least five distinct types of dentate neuron terminating in mutually exclusive domains of the cell's surface in four out of five cases. Four of the cell types (HICAP cell, MOPP cell, basket cell, axo-axonic cell) contain GABA, and the HIPP cell may also be inhibitory. The specific local inhibitory neurons terminating in conjunction with particular excitatory amino acid inputs to the granule cells (types 1 – 3) are in a position to interact selectively with the specific inputs on the same dendritic segment. This arrangement provides a possibility for the independent regulation of the gain and long-term potentiation of separate excitatory inputs, through different sets of GABAergic local circuit neurons. The pairing of excitatory and inhibitory inputs may also provide a mechanism for the downward reseating of excitatory postsynaptic potentials, thereby extending their dynamic range.     

Tuning afferent synapses of hippocampal interneurons by neuropeptide Y

Cholecystokinin (CCK)‐expressing basket cells encompass a subclass of inhibitory GABAergic interneurons that regulate memory‐forming oscillatory network activity of the hippocampal formation in accordance to the emotional and motivational state of the animal, conveyed onto these cells by respective extrahippocampal afferents. Various excitatory and inhibitory afferent and efferent synapses of the hippocampal CCK basket cells express serotoninergic, cholinergic, cannabinoid, and benzodiazepine sensitive receptors, all contributing to their functional plasticity. We explored whether CCK basket cells are modulated by neuropeptide Y (NPY), one of the major local neuropeptides that strongly inhibits hippocampal excitability and has significant effect on its memory function. Here, using GAD65‐GFP transgenic mice for prospective identification of CCK basket cells and whole‐cell patch‐clamp recordings, we show for the first time that excitatory and inhibitory inputs onto CCK basket cells in the dentate gyrus of the hippocampus are modulated by NPY through activation of NPY Y2 receptors. The frequency of spontaneous and miniature EPSCs, as well as the amplitudes of stimulation‐evoked EPSCs were decreased. Similarly, the frequency of both spontaneous and miniature IPSCs, and the amplitudes of stimulation‐evoked IPSCs were decreased after NPY application. Most of the effects of NPY could be attributed to a presynaptic site of action. Our data provide the first evidence that the excitatory and inhibitory inputs onto the CCK basket cells could be modulated by local levels of NPY, and may change the way these cells process extrahippocampal afferent information, influencing hippocampal function and its network excitability during normal and pathological oscillatory activities. © 2009 Wiley‐Liss, Inc.     

Rearrangement of synaptic connections with inhibitory neurons in developing mouse visual cortex

Cortical inhibition is determined in part by the organization of synaptic inputs to gamma-aminobutyric acidergic (GABAergic) neurons. In adult rat visual cortex, feedforward (FF) and feedback (FB) connections that link lower with higher areas provide approximately 10% of inputs to parvalbumin (PV)-expressing GABAergic neurons and approximately 90% to non-GABAergic cells (Gonchar and Burkhalter [1999] J. Comp. Neurol. 406:346-360). Although the proportions of these targets are similar in both pathways, FF synapses prefer larger PV dendrites than FB synapses, which may result in stronger inhibition in the FF than in the FB pathway (Gonchar and Burkhalter [1999] J. Comp. Neurol. 406:346-360). To determine when during postnatal (P) development FF and FB inputs to PV and non-PV neurons acquire mature proportions, and whether the pathway-specific distributions of FF and FB inputs to PV dendrites develop from a similar pattern, we studied FF and FB connections between area 17 and the higher order lateromedial area (LM) in visual cortex of P15-42 mice. We found that the innervation ratio of PV and non-PV neurons is mature at P15. Furthermore, the size distributions of PV dendrites contacted by FF and FB synapses were similar at P15 but changed during the third to sixth postnatal weeks so that, by P36-42, FF inputs preferred thick dendrites and FB synapses favored thin PV dendrites. These results suggest that distinct FF and FB circuits develop after eye opening by rearranging the distribution of excitatory synaptic inputs on the dendritic tree of PV neurons. The purpose of this transformation may be to adjust differentially the strengths of inhibition in FF and FB circuits.     

Gap junctions linking the dendritic network of GABAergic interneurons in the hippocampus.

The network of GABAergic interneurons connected by chemical synapses is a candidate for the generator of synchronized oscillations in the hippocampus. We present evidence that parvalbumin (PV)-containing GABAergic neurons in the rat hippocampal CA1 region, known to form a network by mutual synaptic contacts, also form another network connected by dendrodendritic gap junctions. Distal dendrites of PV neurons run parallel to the alveus (hippocampal white matter) and establish multiple contacts with one another at the border between the stratum oriens and the alveus. In electron microscopic serial section analysis, gap junctions could be identified clearly at 24% of these contact sites. A dendrodendritic chemical synapse and a mixed synapse also were found between PV-immunoreactive dendrites. Three-dimensional reconstruction of the dendritic arborization revealed that both PV neurons of the well known vertical type (presumptive basket cells and axoaxonic cells) and those of another horizontal type constitute the dendritic network at the light microscopic level. The extent of dendritic fields of single PV neurons in the lateral direction was 538 +/- 201 micrometer (n = 5) in the vertical type and 838 +/- 159 micrometer (n = 6) in the horizontal type. Our previous and present observations indicate that PV-containing GABAergic neurons in the hippocampus form the dual networks connected by chemical and electrical synapses located at axosomatic and dendrodendritic contact sites, respectively. Gap junctions linking the dendritic network may mediate coherent synaptic inputs to distant interneurons and thereby facilitate the synchronization of oscillatory activities generated in the interneuron network.     

Ultrastructure of commissural neurons of the hilar region in the hippocampal dentate gyrus

Previous studies have described the polymorph neurons in the hilus of the dentate gyrus at the light microscopic level and have indicated that many of those neurons are the cells of origin for both ipsilateral associational and commissural projections to the dentate gyrus. Because previous studies have not described the ultrastructural characteristics of the hilar neurons, we identified these features of the commissural neurons in the hilus. The method of retrograde transport of horseradish peroxidase (HRP) was utilized with a silver staining technique for HRP intensification. Two populations of labeled commissural neurons were observed in electron microscopic preparations of the contralateral hilus. One type consisted of cells with somata that exhibited round or oval nuclei with no intranuclear inclusions and formed symmetric axosomatic synapses. The main dendrites of those neurons were thick and tapering. In contrast, the other type of labeled neuronal soma had infolded nuclei containing intranuclear rods or sheets, displayed both symmetric and asymmetric axosomatic synapses, and had dendrites that were less thick and generally aspinous. In those same preparations, labeled commissural axon terminals formed synapses with dendrites and dendritic spines in the hilus and molecular layer and iwth somata in the granule cell layer. From the results of this study it appears that there are two distinct populations of commissural hilar neurons: one type resembles the morphology of the spiny CA3 pyramidal neuron, a type of excitatory projection cell, and the other type is similar to the dentate gyrus basket cell, a local circuit neuron associated with GABAergic inhibition. This latter cell type provides further support for the notion that some commissural neurons are inhibitory.     

Parvalbumin‐producing striatal interneurons receive excitatory inputs onto proximal dendrites from the motor thalamus in male mice

In rodents, the dorsolateral striatum regulates voluntary movement by integrating excitatory inputs from the motor‐related cerebral cortex and thalamus to produce contingent inhibitory output to other basal ganglia nuclei. Striatal parvalbumin (PV)‐producing interneurons receiving this excitatory input then inhibit medium spiny neurons (MSNs) and modify their outputs. To understand basal ganglia function in motor control, it is important to reveal the precise synaptic organization of motor‐related cortical and thalamic inputs to striatal PV interneurons. To examine which domains of the PV neurons receive these excitatory inputs, we used male bacterial artificial chromosome transgenic mice expressing somatodendritic membrane–targeted green fluorescent protein in PV neurons. An anterograde tracing study with the adeno‐associated virus vector combined with immunodetection of pre‐ and postsynaptic markers visualized the distribution of the excitatory appositions on PV dendrites. Statistical analysis revealed that the density of thalamostriatal appositions along the dendrites was significantly higher on the proximal than distal dendrites. In contrast, there was no positional preference in the density of appositions from axons of the dorsofrontal cortex. Population observations of thalamostriatal and corticostriatal appositions by immunohistochemistry for pathway‐specific vesicular glutamate transporters confirmed that thalamic inputs preferentially, and cortical ones less preferentially, made apposition on proximal dendrites of PV neurons. This axodendritic organization suggests that PV neurons produce fast and reliable inhibition of MSNs in response to thalamic inputs and process excitatory inputs from motor cortices locally and plastically, possibly together with other GABAergic and dopaminergic dendritic inputs, to modulate MSN inhibition.     

Afferent and efferent synaptic connections of somatostatin-immunoreactive neurons in the rat fascia dentata

The aim of this study was to determine whether somatostatin (SS)-immunoreactive neurons of the rat fascia dentata are involved in specific excitatory circuitries that may result in their selective damage in models of epilepsy. Synaptic connections of SS-immunoreactive neurons were determined at the electron microscopic level by using normal and colchicine pretreated rats. Vibratome sections prepared from both fascia dentata of control animals and from rats that had received an ipsilateral lesion of the entorhinal cortex 30-36 hours before sacrifice were immunostained for SS by using a monoclonal antibody (SS8). Correlated light and electron microscopic analysis demonstrated that many SS-immunoreactive neurons in the hilus send dendritic processes into the outer molecular layer of the fascia dentata, and dendrites of the same neurons occupy broad areas in the dentate hilar area. The majority of SS-immunoreactive axon terminals form symmetric synapses with the granule cell dendrites in the outer molecular layer and also innervate deep hilar neurons. Via their dendrites in the outer molecular layer, the SS-immunoreactive neurons receive synaptic inputs from perforant pathway axons which were identified by their anterograde degeneration following entorhinal lesions. The axons from the entorhinal cortex are the first segment of the main hippocampal excitatory loop. The hilar dendrites of the same SS-immunoreactive cells establish synapses with the mossy axon collaterals which represent the second member in this excitatory neuronal chain. These observations suggest that SS-immunoreactive neurons in the dentate hilar area may be driven directly by their perforant path synapses and via the granule cells which are known to receive a dense innervation from the entorhinal cortex. These observations demonstrate that SS-immunoreactive neurons in the hilar region are integrated in the main excitatory impulse flow of the hippocampal formation.     

Input-dependent synaptic targeting of alpha(2)-subunit-containing GABA(A) receptors in synapses of hippocampal pyramidal cells of the rat

Pyramidal cells, expressing at least 14 subunits of the heteropentameric GABA(A) receptor, receive GABAergic input on their soma and proximal dendrites from basket cells, activating GABA(A) receptors and containing either parvalbumin or cholecystokinin and vasoactive intestinal polypeptide. The properties of GABA(A) receptors are determined by the subunit composition, and synaptic receptor content governs the effect of the presynaptic neuron. Using a quantitative electron microscopic immunogold technique, we tested whether the synapses formed by the two types of basket cell show a difference in the subunit composition of GABA(A) receptors. Terminals of one of the basket cells were identified by antibodies to parvalbumin. Synapses made by parvalbumin-negative terminals showed five times more immunoreactivity for the alpha(2) subunit than synapses made by parvalbumin-positive basket cells, whose synapses were frequently immunonegative. This difference is likely to be due to specific GABA(A) receptor alpha subunit composition, because neither synaptic size nor immunoreactivity for the beta(2/3) subunits, indicating total receptor content, was different in these two synapse populations. Synapses established by axo-axonic cells on axon initial segments showed an intermediate number of immunoparticles for the alpha(2) subunit compared to those made by basket cells but, due to their smaller size, the density of the alpha(2) subunit immunoreactivity was higher in synapses on the axon. Because the two basket cell types innervate the same domain of the pyramidal cell, the results indicate that pyramidal cells have mechanisms to target GABA(A) receptors, under presynaptic influence, preferentially to distinct synapses. The two basket cell types act via partially distinct GABA(A) receptor populations.     

Subcellular distribution of high-voltage-activated calcium channel subtypes in rat globus pallidus neurons.

Globus pallidus (GP) neurons receive dense inhibitory synaptic inputs interspersed with sparse excitatory inputs distributed across the entire extent of their somata and dendrites. Yet, despite this predominance of inhibitory influence, GP neurons fire at a high tonic rate, suggesting that intrinsic properties play an important role in determining the physiological characteristics of these neurons. High-voltage-activated (HVA) calcium channels represent an important class of conductances that plays roles in controlling neurotransmitter release, postsynaptic excitability, and intracellular calcium signaling. To better understand the intrinsic properties of GP neurons, we examined the subcellular localization of HVA calcium channels by using immunocytochemistry at the electron microscopic level. Peroxidase labeling with antibodies against P/Q-, N-, and R-type HVA calcium channels demonstrated the presence of these channels in both proximal and distal dendrites of GP neurons. P/Q-, N-, and R-type channels were also found in presynaptic terminals, whereas L-type channels were found exclusively postsynaptically in neuronal elements. Immunogold labeling demonstrated that, although the density of intracellular L-type calcium channel labeling remains constant throughout the proximal-distal extent of the dendritic tree of GP neurons, the density of plasma membrane-bound channels is greater in distal dendrites. The finding of HVA calcium channels distributed throughout the whole dendritic tree of GP neurons indicates that these channels may interact with synaptic inputs to allow rich processing possibilities for GP neuron dendrites. Furthermore, the finding of a greater density of plasma membrane-bound L-type channels in distal dendrites expands the view that L-type channels are important only in somatic and proximal locations.     

Morphological characteristics of mouse stellate and basket cells and their neuroglial envelope: an electron microscopic study

Sampling of stellate cells from the molecular layer of cerebellar cortex at two distinct levels within yielded two populations of cells referred to as stellate and basket cells respectively. The appearance of the soma, the several kinds of dendrites and their accompanying spines can be characterized on the electron microscopic level for each type of cell and correlate well with Golgi impregnation studies. Analytical studies were made to determine the proportion of neural and glial elements in direct apposition to stellate and basket soma and their respective dendrites. On the average the glial covering of a basket or stellate cell was 12–14% of the cell profile, reaching 18.6% for the distal portions of dendrites. A quantitative analysis of the amount of soma and dendritic perimeter in contact with synaptic boutons revealed the percentage of perimeter surface covered by terminal boutons was 15.2% for basket soma, 4.5% for the stellate soma, 21% for basket dendrites and 10.3% for stellate dendrites. An interesting group of cells located in the Purkinje cell layer had the morphological characteristics of basket cells but an unusual synaptic input and an unusually high proportion of glial envelope due to their unique position. This report is an introduction to a comparative analysis of the distribution, and densities of different types of synapses upon basket and stellate cells.     

Terminal field of cholecystokinin-8-like immunoreactive projection neurons of the rat main olfactory bulb

The terminal field of cholecystokinin-8 (CCK)-like immunoreactive (CCK-IR) tufted cells in the rat main olfactory bulb was examined by means of immunohistochemistry combined with either an anterograde tracer or a degeneration method. CCK immunostaining was carried out in animals in which Phaseolus vulgaris agglutinin (PHA) had been injected into the main olfactory bulb. Pairs of adjacent sections were processed for CCK and PHA immunostaining, respectively. Dense CCK-IR terminallike staining was noted in layer Ia of the anterior olfactory nucleus and lateral part of the olfactory tubercle; weaker staining was also observed in the transitional area between the anterior olfactory nucleus and the piriform cortex, in the medial part of the olfactory tubercle, and in the cortical amygdaloid nucleus. The CCK-IR staining was limited to the area containing PHA-labeled terminals and was diminished in these sites after unilateral olfactory bulbectomy. Immuno-electron microscopic analysis showed that CCK-IR profiles in such regions made asymmetric synaptic contacts, mainly with dendritic spines. These results suggest that CCK-IR tufted cells project mainly to the anterior olfactory nucleus and lateral part of the olfactory tubercle, and act mainly via axospinous synapses.     

Morphology, Electrophysiology and Pathophysiology of Supragranular Neurons in Rat Primary Somatosensory Cortex

Intracellularly biocytin-labelled neurons in layers 11/111 of adult rat primary somatosensory cortex were analysed for their morphological and electrophysiological properties and studied for their response pattern to transient hypoxia under in vitro conditions. The largest dendritic region is formed by the basal dendrites, which constitute an average area of 0.06 mm² and which can receive synaptic inputs over horizontal distances of more than 300 μm. The dendritic territories formed by the oblique dendrites situated on the apical trunk and by the apical tuft are much smaller. The spine density is highest on the apical trunk, suggesting that large numbers of excitatory synapses are present in this region of the cell. All neurons revealed intrinsic membrane properties of typical regular spiking cells and received an excitatory and a strong biphasic inhibitory input. Whereas a significant correlation could be detected between the cell's input resistance and soma area, no correlation existed between the cell's total dendritic length and input resistance or membrane time constant/input resistance. Neurons responded to transient hypoxia either with an anoxic hyperpolarization with an apparent reversal potential of -82.4 mV, or with a gradual anoxic depolarization which reversed at -56 mV. Oxygen deprivation caused a significant reduction in the extent of axonal collaterals, whereas dendritic proportions and spine density were unaffected. The present study indicates that the dendritic tree is well preserved under in vitro conditions, whereas axonal connections are diminished by oxygen deprivation. Our results further suggest that certain structural properties correlate with the cellular physiology, but that the cell's morphology does not determine its responsiveness to hypoxia.     

A bushy cell network in the rat ventral cochlear nucleus

Geometry of the dendritic tree and synaptic organization of afferent inputs are essential factors in determining how synaptic input is integrated by neurons. This information remains elusive for one of the first brainstem neurons involved in processing of the primary auditory signal from the ear, the bushy cells (BCs) of the ventral cochlear nucleus (VCN). Here, we labeled the BC dendritic trees with retrograde tracing techniques to analyze their geometry and synaptic organization after immunofluorescence for excitatory and inhibitory synaptic markers, electron microscopy, morphometry, double tract‐tracing methods, and 3D reconstructions. Our study revealed that BC dendrites provide space for a large number of compartmentalized excitatory and inhibitory synaptic interactions. The dendritic inputs on BCs are of cochlear and noncochlear origin, and their proportion and distribution are dependent on the branching pattern and orientation of the dendritic tree in the VCN. Three‐dimensional reconstructions showed that BC dendrites branch and cluster with those of other BCs in the core of the VCN. Within the cluster, incoming synaptic inputs establish divergent multiple‐contact synapses (dyads and triads) between BCs. Furthermore, neuron–neuron connections including puncta adherentia, sarcoplasmic junctions, and gap junctions are common between BCs, which suggests that these neurons are electrically coupled. Overall, our study demonstrates the existence of a BC network in the rat VCN. This network may establish the neuroanatomical basis for acoustic information processing by individual BCs as well as for enhanced synchronization of the output signal of the VCN. J. Comp. Neurol. 516:241–263, 2009. © 2009 Wiley‐Liss, Inc.