Non-pyramidal Neurons In Layers Research Articles

Kv2 channels regulate firing rate in pyramidal neurons from rat sensorimotor cortex

The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7-P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold 'bullets' coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non-transfected cell from the same layer and area. Whole-cell voltage-clamp recordings obtained after 3-7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from -78 to -2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current-clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady-state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na(+) channels from inactivation. Depolarization and the number of inactivated Na(+) channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.

A cross-study meta-analysis and three-dimensional comparison of cell counting in the anterior cingulate cortex of schizophrenic and bipolar brain

Using a two-dimensional cell counting approach, a 1991 study in the anterior cingulate cortex (ACCx) detected a reduction in the density of nonpyramidal neurons in layers II–VI of schizophrenic subjects. Schizophrenics without superimposed mood disturbances showed a 16% decrease in layer II, while schizoaffectives showed a 30% decrease, suggesting that a decreased density of nonpyramidal neurons in layer II of ACCx might vary more strongly with affective disorder than with schizophrenia. Two follow-up studies from this laboratory, one a replication of that reported in 1991 and the other an analysis of tyrosine hydroxylase immunoreactive fibers, were undertaken in ACCx of normal controls and schizophrenics. These three data sets have been combined and a meta-analysis of the density of pyramidal, nonpyramidal and glial cells was performed to explore whether changes in the density of interneurons in ACCx may be a reliable finding in the major psychoses. Not all groups have reported this finding, but several had employed a different cell counting technique (i.e. three dimensional optical dissector), which could help to explain the discrepant findings in schizophrenia and affective disorder. The data from each of three different studies (now designated as studies A, B and C, respectively) have been internally normalized, combined into a single dataset and analyzed using nonparametric statistics. Tissue blocks from a subset of cases in study B (six controls, six schizophrenics and six bipolars) were embedded in celloidin and counted using an “unbiased” three dimensional counting method (study D). The data from studies A and B indicate that the density of nonpyramidal neurons in layer II of ACCx in the schizoaffective and bipolar samples was significantly decreased. In the schizophrenics, the nonpyramidal neurons were also decreased, but only by 15%. All three groups also showed a decrease of pyramidal neurons in layers IV, V and VI, but this difference was significant only in layer IV of the schizophrenics. When data from study C were added, the differences in pyramidal and nonpyramidal neurons were less striking. For study D, the pattern of findings are strikingly similar to those obtained in studies A, B and C, indicating that both 2D and 3D cell counting methodologies are capable of detecting the same differences. Taken together, these results indicate that the earlier finding of a decreased density of nonpyramidal neurons in ACCx of schizophrenics is consistent across non-overlapping subjects and/or methods in four separate studies, and is more pronounced in schizoaffective and bipolar subjects than in schizophrenics without superimposed mood disturbance.

Rapid arrival and integration of ascending sensory information in layer 1 nonpyramidal neurons and tuft dendrites of layer 5 pyramidal neurons of the neocortex.

Ascending sensory inputs arriving in layer 1 of the neocortex carry crucial signals for detecting salient information; but how the inputs are processed in layer 1 is unknown. Using a whole-cell in vivo recording technique targeting nonpyramidal neurons in layer 1 and tuft dendrites of layer 5 pyramidal neurons in layers 1-2, we examined the processing of these ascending sensory inputs in the barrel cortex. Here, we show that local circuit and deeper-layer-projecting neurons in layer 1, as well as tuft dendrites and somata of layer 5 pyramidal neurons, respond to multiple whiskers (6-15) with robust EPSPs. Remarkably, the latency for primary whisker-evoked responses is as short as approximately 5-7 msec in layer 1 neurons and tuft dendrites of layer 5 pyramidal neurons. In addition, the latency for primary whisker-evoked responses in tuft dendrites of layer 5 pyramidal neurons is approximately 1 msec shorter than that in somata. These results indicate that ascending sensory inputs arrive in layers 1 and 4 concurrently, which provides a neural mechanism for rapid integration and coincident detection of salient sensory information.

Parvalbumin neuron circuits and microglia in three dopamine-poor cortical regions remain sensitive to amphetamine exposure in the absence of hyperthermia, seizure and stroke

The dopamine-releasing and depleting substance amphetamine (AMPH) can make cortical neurons susceptible to damage, and the prevention of hyperthermia, seizures and stroke is thought to block these effects. Here we report a 2-day AMPH treatment paradigm which affected only interneurons in three cortical regions with average or below-average dopamine input. AMPH (six escalating doses/day ranging from 5 to 30 mg/kg for 2 days) was given at 17–18 °C ambient temperature (T) to adult male rats. During the 2-day AMPH treatment, peak body T stayed below 38.9 °C in 40% of the AMPH treated rats. In 60% of the rats, deliberate cooling suppressed (<39.5 °C) or minimized (<40.0 °C) hyperthermia. Escalation of stereotypes to seizure-like behaviors was rare and post-mortem morphological signs of stroke were absent. Neurons labeled with the anionic, neurodegeneration-marker dye Fluoro-Jade (F-J) were seen 1 day after dosing, peaked 3 days later, but were barely detectable 14 days after dosing. Only nonpyramidal neurons in layer IV of the somatosensory barrel cortex and in layer II of the piriform cortex and posterolateral cortical amygdaloid nucleus were labeled with Fluoro-Jade. Isolectin B-labeled activated microglia were only detected in their neighborhood. F-J labeled neurons were extremely rare in cortical regions rich in dopamine (e.g. cingulate cortex), and were absent in cortical regions with no dopamine (e.g. visual cortex). Parvalbumin was seen in some Fluoro-Jade-labeled neurons and parvalbumin immunostaining in local axon plexuses intensified. This AMPH paradigm affected fewer cortical regions, and caused smaller reduction in striatal tyrosine hydroxylase (TH) immunoreactivity than previous 1-day AMPH regimens generating seizures or severe (above 40 °C) hyperthermia. Correlation between peak or mean body T and the extent of neurodegeneration or microgliosis was below statistical significance. Astrogliosis (elevated levels of the astroglia-marker, glial fibrillary acidic protein (GFAP)) was detected in many brain regions. In the striatum and midbrain, F-J labeled neurons and activated microglia were absent, but astrogliosis, decreased TH immunolabel, and swollen TH fibers were detected. In sum, after this AMPH treatment, cortical pyramidal neurons were spared, but astrogliosis was brain-wide and some interneurons and microglia in three cortical regions with average or below-average dopamine input remained sensitive to AMPH exposure.

Developmental changes in calpain activity, GluR1 receptors and in the effect of kainic acid treatment in rat brain

The cellular distribution of calpain activation and glutamate receptor 1 (GluR1) subunits of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and their alterations following kainic acid-induced seizure were evaluated during postnatal development using antibodies specific for spectrin breakdown product and the C-terminus of GluR1 subunits. In the first postnatal week, most brain regions exhibited high levels of calpain activity that progressively decreased during the following weeks. The highest levels of spectrin breakdown product immunoreactivity were observed in the somata and proximal dendrites of hippocampal pyramidal cells, non-pyramidal neurons in stratum oriens, and cortical neurons. In general, during the first two postnatal weeks, kainic acid treatment induced a decrease in spectrin breakdown product immunoreactivity in neuronal cell bodies and an increase in dendritic fields. Obvious elevation in spectrin breakdown product immunoreactivity in selective non-pyramidal cells in stratum oriens started at postnatal day 14, and was further evidenced by postnatal day 21. Likewise, massive calpain activation in subpopulations of neurons in some thalamic nuclei, amygdala, and pyriform cortex was observed after the third postnatal week. GluR1 subunits were highly expressed throughout the forebrain in the first postnatal week, further increased during the second postnatal week, decreased thereafter, and reached adult levels after postnatal day 21. In cortex, intense GluR1 immunostaining was found in the somata and proximal processes of pyramidal and non-pyramidal neurons, with the non-pyramidal neurons in layers IV through VI exhibiting the densest immunolabelling. In the first two postnatal weeks, the somata of hippocampal pyramidal neurons exhibited intense GluR1 immunostaining that became more dendritic in the subsequent developmental period. While hilar cells exhibited a similar developmental pattern as CA regions, the molecular layer of dentate gyrus exhibited weak immunoreactivity from postnatal day 7 to postnatal day 14. The early increase in GluR1 immunoreactivity in hippocampal pyramidal layer following kainic acid treatment occurred throughout the developmental period, while the later decrease in CA regions, amygdala, and pyriform cortex was observed only in postnatal day 21 animals. The combined immunocytochemical studies of spectrin breakdown product localization and GluR1 expression indicate that calpain activation might play an important role in synaptic formation, developmental regulation of synaptic plasticity, and neuronal vulnerability to excitotoxicity during postnatal development. Moreover, calpain-mediated modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors might underlie these processes.

Quantitative Three-Dimensional Analysis of the Catecholaminergic Innervation of Identified Neurons in the Macaque Prefrontal Cortex

The present study provides a complete quantitative three-dimensional analysis of neurons in primate prefrontal cortex targeted by catecholaminergic axons. Individual pyramidal and nonpyramidal cells in fixed slices were filled with Lucifer yellow (LY) and recovered with anti-LY antibody combined with anti-tyrosine hydroxylase (TH) antisera to reveal catecholaminergic axons. The total number of TH contacts and TH apposition density (THAD) was obtained for pyramidal and nonpyramidal cells in different layers. Four TH contacts (two on spines and two on shafts) were selected for correlated electron microscopic examination and serially sectioned; all four were confirmed as membrane appositions. Quantitative analysis revealed 90 TH contacts per pyramidal neuron in layer III, with a density of 0.8 per 100 microm of dendritic length (i.e., averaging one contact per basal dendrite). Remarkably, pyramids of layers III, V, and VI had the same THAD values, with a highly regular distribution of TH terminals on their spiny dendritic trees. In contrast, TH contacts on nonpyramidal neurons in layer III were half as dense and, moreover, were distributed irregularly and showed large variation from cell to cell. Neurons in layers II and superficial III had the highest THAD, as compared with deeper layers (1.4 vs 0.7 per 100 micron of dendritic length for pyramids; 0.53 vs 0.4 for interneurons). The highly organized TH innervation of pyramidal neurons, with at least one contact on virtually every dendrite, indicates that catecholaminergic, presumably dopaminergic, terminals are placed strategically along the entire dendritic tree to modulate most, if not all, of the excitatory input of a neuron. At the same time, the sparsity of contacts per dendrite may explain cortical vulnerability in diseases involving dopamine.

Dendritic morphology in epileptogenic cortex from TRPE patients, revealed by intracellular Lucifer Yellow microinjection and confocal laser scanning microscopy

Biopsy material was obtained from cortical epileptogenic zones (eight temporal, one occipital, one parietal and one frontal) of eleven patients aged 1.5–47 years with therapy-resistant partial epilepsy (TRPE) undergoing epilepsy surgery. Control autopsy material (two temporal, two occipital, one parietal and one frontal) was removed from six neurologically healthy cases within 6–10 hours postmortem delay. In each specimen, 100–300 pyramidal and non-pyramidal neurons were visualized by intracellular Lucifer Yellow microinjection. Single neurons were imaged using CLSM generated serial optical sections; 2-D reconstruction of each neuron was made using z-projection of serial optical images, and 3-D reconstructions and rotations were computerized. Neuronal maps from TRPE biopsies, compared to control autopsies, show markedly increased numbers of dendritic abnormalities of single pyramidal and non-pyramidal neurons in layers I, II-III, V-VII, and in the subcortical white matter. The abnormalities include: 1. (1) increased number of non-pyramidal cells in layer I 2. (2) many pyramidal cells with two or three dendrites originating apically, rather than one single apical dendrite, in layers II-III 3. (3) atypical orientation of oblique apical and basal dendrites in pyramidal neurons of layers II-VII 4. (4) increased number of atypical ‘dinosaur-like’ and fusiform cells in layers V-VII 5. (5) numerous neurons in the white matter. These abnormalities may be etiological in cases with early onset, and predisposing in cases with late onset.

Co-localization of two calcium binding proteins in GABA cells of rat piriform cortex

In the rat piriform cortex, double-fluorescence immunocytochemistry was used to study the coexistence of two calcium binding proteins, parvalbumin and calbindin D28k, and GABA. Layer I cells were immunoreactive only for GABA. In layer II a small number of cells were immunoreactive for all three substances. A few other cells contained only parvalbumin and GABA immunoreactivity. In layer III a subpopulation of calbindin D28k immunoreactive cells (about 60%) were also immunoreactive for both parvalbumin and GABA. The remainder showed only calbindin D28k and GABA immunoreactivity. Many immunoreactive terminals, which surrounded non-immunoreactive cell somata to form basket endings in layers II and III, were immunoreactive for all three substances. Their major source may be the non-pyramidal neurons in layer III, which contained the three chemical substances.

Heterogeneity of layer II neurons in human entorhinal cortex.

Abnormalities in the layer II neurons of human entorhinal cortex have been implicated in the pathophysiology of Alzheimer's disease and schizophrenia. The reported abnormalities are not homogeneously distributed throughout the entorhinal cortex, suggesting that layer II of entorhinal cortex may contain different subpopulations of neurons, each with a different susceptibility to pathological mechanisms. In order to investigate the possible heterogeneity of neurons in layer II of human entorhinal cortex, we first identified distinct subdivisions of human entorhinal cortex by adapting the cytoarchitectonic criteria for subdivisions of monkey entorhinal cortex described by Amaral et al. (J Comp Neurol 264:326, 1987). The morphology and regional distribution of distinct subpopulations of human layer II neurons were determined through the use of immunohistochemical techniques. Multipolar, stellate, and modified pyramidal neurons in the characteristic cell clusters or islands of layer II were immunoreactive for nonphosphorylated neurofilament proteins. The intensity of immunoreactivity for the nonphosphorylated neurofilament proteins gradually increased along the rostrocaudal axis of entorhinal cortex and was primarily due to a similar gradient in the density of labeled neurons per island. The calcium-binding protein calbindin D-28K was found in both pyramidal and nonpyramidal neurons in layers II and superficial III. The distribution of calbindin-immunoreactive neurons also depended upon the region of entorhinal cortex. In rostral entorhinal cortex, labeled neurons were scattered throughout the superficial layers, whereas in caudal entorhinal cortex, distinctive patches of small calbindin-immunoreactive neurons were found among the layer II islands. Another calcium-binding protein, parvalbumin, was present in nonpyramidal neurons in layers II and III that were distinct from those containing calbindin. The regional distribution of parvalbumin-positive neurons was very similar to that of the neurofilament immunoreactive neurons; in rostral entorhinal cortex very few parvalbumin-labeled neurons were present but their frequency gradually increased in the caudal direction. In addition, punctate parvalbumin immunoreactivity was frequently encountered in the location of the nonphosphorylated neurofilament protein-positive layer II islands. These findings demonstrate that layer II of human entorhinal cortex contains distinct subpopulations of neurons, that the relative density of each subpopulation differs across cytoarchitectonic regions, and that the patterns of distribution of these subpopulations are in some cases similar and in other cases complementary. This heterogeneity in the organization of layer II of human entorhinal cortex has important implications for the study of some neuropsychiatric disorders.

Dementia of frontal lobe type and amyotrophy.

Dementia of frontal lobe type may precede motor signs in a number of adult patients with amyotrophy. Neuropathological studies have shown neuron loss, spongiosis and gliosis mainly in layers II and III of the frontal and temporal lobes, together with myelin pallor of the subcortical white matter. Golgi studies revealed loss of dendritic spines on the apical dendrite of layer III pyramidal neurons, decreased numbers of dendrites, amputation and tortuosities of dendrites, and distal and proximal dendritic swellings and enlargements. Calbindin D-28K immunocytochemistry revealed a marked decrease in the number of cortical immunoreactive neurons and loss of immunoreactivity in dendrites of the remaining cells. These features indicate that pyramidal and non-pyramidal neurons in layers II and III are severely damaged, and suggest that cortical processing is seriously impaired in patients with frontal lobe type dementia.

Dementia of Frontal Lobe Type and Amyotrophy

Dementia of frontal lobe type may precede motor signs in a number of adult patients with amyotrophy. Neuropathological studies have shown neuron loss, spongiosis and gliosis mainly in layers II and III of the frontal and temporal lobes, together with myelin pallor of the subcortical white matter. Golgi studies revealed loss of dendritic spines on the apical dendrite of layer III pyramidal neurons, decreased numbers of dendrites, amputation and tortuosities of dendrites, and distal and proximal dendritic swellings and enlargements. Calbindin D-28K immunocytochemistry revealed a marked decrease in the number of cortical immunoreactive neurons and loss of immunoreactivity in dendrites of the remaining cells. These features indicate that pyramidal and non-pyramidal neurons in layers II and III are severely damaged, and suggest that cortical processing is seriously impaired in patients with frontal lobe type dementia.

Distribution of neurons in the pigmented rabbit's visual cortex

Past observations revealed a relationship between mammalian retinal ganglion cell distribution and cortical and subcortical projection of the rabbit's visual field (3, 4, 8). High ganglion cell density sectors of the retina claimed larger areas in their cortical and subcortical projections (with a high magnification factor) than retinal areas with low ganglion cell density. In this study, it was observed that distribution of nonpyramidal neurons in laminae IV and VI of the pigmented rabbit's visual cortex was not uniform. The nonuniformity in layer IV roughly followed the variation in the magnification factor in the cortical projection of the animal's visual field. The highest density of nonpyramidal neurons in layer IV was observed in the cortical area with the highest magnification factor.

Anatomical and physiological properties of the projection from the sensory cortex to the motor cortex in normal cats: the difference between corticocortical and thalamocortical projections

Details of the distribution of terminal sites of the projection fibers from area 2 of the sensory cortex to the motor cortex were studied and compared with the distribution of terminals from the ventrolateral (VL) nucleus of the thalamus to the motor cortex. The results obtained were as follows: (1) Intracortical microstimulation (ICMS) in area 2 produced measurable short-latency EPSPs only in neurons located in layers II and III of the motor cortex, whereas VL stimulation produced short-latency EPSPs in neurons throughout the depths of the motor cortex. (2) The time from the beginning to the peak of the EPSPs was not significantly different for area 2- and VL-elicited EPSPs suggesting that there was no systematic difference between effective terminal sites for both inputs. However, there was a difference when a given neuron received both inputs suggesting that there was a segregation between the two inputs within a given cell. (3) The majority of area 2-elicited EPSPs were smooth and monophasic, but some (40%) of them showed double peaks indicating that some neurons received mono- and disynaptic inputs from area 2. (4) Intracellular injections of HRP suggested that neurons receiving input from area 2 were predominantly multipolar non-pyramidal neurons in layers II and III whereas neurons receiving thalamic input were pyramidal as well as non-pyramidal cells. (5) Field potentials in the motor cortex evoked by area 2 stimulation did not change polarity in the depths of the cortex and therefore, differed from the VL-evoked potentials suggesting differences in the mechanisms of generating the electrical fields. (6) It is concluded that association fibers effective for producing EPSPs terminate primarily on non-pyramidal cells in layer II and III whereas VL fibers terminate not only on pyramidal but also on non-pyramidal cells in layers III and V. This study provided a basis for examining the modifiability of association fibers after elimination of VL input to the motor cortex which is reported in the following paper.

The non-pyramidal cells in layer III of cat primary auditory cortex (AI).

The form and location of non-pyramidal neurons in layer III of the primary auditory cortex (AI) of adult cats is described in Golgi, Nissl, and other material. The cells were compared to the profiles of retrogradely labeled, commissurally interconnected cells. A principal finding is that certain non-pyramidal and pyramidal cells project interhemispherically to AI; a second conclusion is that the retrogradely labeled commissural cells form small clusters or narrow strips separated by unlabeled patches even after massive injections in the opposite AI. The non-pyramidal cells of origin have not yet been conclusively identified, but they must include one (or more) of the following six types of cells observed in Golgi-impregnated material: tufted or bitufted cells with a radially elongated dendritic arbor; sparsely spinous stellate neurons with thin, smooth dendrites and vertically disposed axonal branches; small stellate cells with varicose dendrites, a restricted dendritic field, and a profusely branched local axon; bipolar neurons with long, thin dendrites; medium-sized multipolar cells with radiating, sparsely branched dendrites; and small stellate neurons with smooth dendrites and a tiny dendritic field. These non-pyramidal cells are found throughout layer III but are more numerous in the upper part, layer IIIa, where they mingle with the small pyramidal neurons. As a rule the axonal branches of non-pyramidal cells are more numerous than those arising from layer III pyramidal neurons, and although they have many axonal collaterals, most project locally and vertically in narrow radial strips. In contrast, pyramidal cell axons have ascending and descending components which invade large, lateral territories in many cortical layers. Layer III non-pyramidal neurons are similar to those in layer IV in certain respects, although their dendritic fields are more spherical and less tufted than those of layer IV cells, and their axons have more local, limited targets. These axons appear to contribute but little to the conspicuous, lateral fiber striae in layer III. The primary intrinsic targets of non-pyramidal cell axons appear to be the apical dendrites of medium-sized and large layer III pyramidal cells, and recurrent branches to the parent cell; their fine, distal branches fortify the vertical plexus in layer III, and certain axons may descend into layer IV. Since layer III in AI receives both commissural and thalamic input, it is possible that these parallel, afferent channels are to some degree segregated, and to some degree convergent, onto particular types of cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The growth of non-pyramidal neurons in the visual cortex of the rat: A morphometric study

In Golgi-Cox preparations, we have studied the 3-dimensional geometry and development of the perikarya and dendrites of non-pyramidal neurons in layer IV of the rat visual cortex with the aid of a semi-automated system. During the first postnatal week, non-pyramidal neurons are sparse and have small perikarya and undifferentiated dendrites. Their number increases substantially during the subsequent few days and reaches adult proportions by the end of the second week of postnatal life. A dramatic increase in the size of neuronal perikarya and elaboration of dendrites occurs from the middle of the first week to the end of the second week. The basic structure of the dendritic fields — as judged by the number of dendritic segments, the length of intermediate and terminal segments, the total length of the dendritic field, and the radial distance of the terminal tips from the center of the cell body — is achieved by the middle of the third postnatal week. The only growth which continues after this stage of development, and at least until day 90, is the elongation of the terminal dendrites. This and other studies indicate that the overall pattern of growth of pyramidal and non-pyramidal neurons is similar, and both cell types reach maturity at approximately the same stage of development. Measurements of non-pyramidal neurons in coronal, sagittal and tangential sections of visual cortex of developing and young-adult rats suggest that the perikarya and dendrites are oriented preferentially in the coronal plane and along an axis extending from the pial surface to the subcortical white matter.