Cortex Pyramidal Neurons Research Articles

Explaining synchrony in feed-forward networks: are McCulloch-Pitts neurons good enough?

In any scientific theory, the conceptual framework already determines the nature and possible scope of the results. Oversimplification prevents an adequate description of the system, whereas too detailed a description obscures the fundamental principles behind the observed phenomena in addition to misspending time and resources. In theoretical neuroscience, this is an important issue because the description level varies widely from detailed biophysical descriptions to abstract computational models. We discuss the question of the appropriate modeling level in the context of a recent report on synchrony in iteratively constructed feed-forward networks of rat cortex pyramidal neuron somata (Reyes, 2003).

Transmitter- and Activity-Dependent Inhibition of Neuronal Firing

Neuronal excitability depends upon the activation of voltage-gated Na + channels. Various neuromodulators reduce Na + channel availability (and thus neuronal excitability) through the activation of heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPCR)-mediated signaling pathways that lead to channel phosphorylation. Carr et al . used a combination of electrophysiological and pharmacological analysis to show that the reduction in Na + channel availability produced by GPCR activation closely resembled slow inactivation--a slow, voltage-dependent state that occurs in response to prolonged depolarization and during repetitive firing. The authors investigated the kinetics and voltage dependence of changes in Na + channel availability in mouse prefrontal cortex pyramidal neurons in response to treatment with the 5-HT 2a/c (5-hydroxytryptamine) receptor agonist 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI). The effects of receptor stimulation were voltage-dependent, and DOI accelerated the development and extent of slow inactivation without affecting the time constant of recovery. Moreover, the effects of DOI, like slow inactivation, were inhibited by increases in the extracellular Na + concentration. Similarly, direct pharmacological activation of protein kinase A and protein kinase C promoted a slow inactivation-like state. The voltage dependence of phosphorylation-dependent channel inactivation occurred after phosphorylation, and mutational analysis indicated that channel phosphorylation was not required for depolarization-dependent development of slow inactivation. DOI accelerated the elevation of threshold that occurs during sustained firing (when the interspike membrane potential remains somewhat depolarized) and led to early failure of action potential generation. Thus, GPCR stimulation, by promoting a slow inactivation-like state, can selectively modulate sustained neuronal activity to produce a novel form of neuronal plasticity. D. B. Carr, M. Day, A. R. Cantrell, J. Held, T. Scheuer, W. A. Catterall, D. J. Surmeier, Transmitter modulation of slow, activity-dependent alterations in sodium channel availability endows neurons with a novel form of cellular plasticity. Neuron 39 , 793-806 (2003). [Online Journal]

The distribution and morphology of prefrontal cortex pyramidal neurons identified using anti-neurofilament antibodies SMI32, N200 and FNP7. Normative data and a comparison in subjects with schizophrenia, bipolar disorder or major depression

Alterations in the density and size of pyramidal neurons in the prefrontal cortex have been described in schizophrenia and mood disorder. However, the changes are generally modest and have not always been replicated. We investigated the possibility that specific pyramidal neuron sub-populations, defined by their immunoreactivity with the anti-neurofilament antibodies SMI32, N200, and FNP7, are differentially affected in these disorders. First, we assessed the distribution and characteristics of pyramidal neurons labelled by the antibodies in the human dorsolateral prefrontal cortex (Brodmann areas 9, 32, 46), using single and double label immunocytochemistry and immunofluorescence. Three largely separate sub-populations of pyramidal neurons were identified, although with more substantial overlap between SMI32- and FNP7-positive neurons in lamina V. We then determined the density, size and shape of the three pyramidal neuron sub-populations in area 9 in patients with schizophrenia, bipolar disorder, or major depressive disorder, compared to controls ( n=15 in each group). We found a lower density of lamina III N200-positive neurons in major depressive disorder than in schizophrenia or bipolar disorder. There were no other overall differences in neuronal density, or in neuronal size or shape, although a planned secondary analysis supported the previously reported decrease of neuronal size in lamina V in bipolar disorder. In summary, our study illustrates a conceptual and methodological approach which may be of value for investigating the differential neuropathological involvement of pyramidal neuron sub-populations. However, we found no clear evidence that the prefrontal neuropathology of schizophrenia or mood disorders preferentially affects SMI32-, N200- or FNP7-immunoreactive pyramidal neurons.

Learning-induced reduction in post-burst after-hyperpolarization (AHP) is mediated by activation of PKC.

We studied the role of protein kinase C (PKC) and protein kinase A (PKA) in mediating learning-related long lasting reduction of the post-burst after-hyperpolarization (AHP) in cortical pyramidal neurons. We have shown previously that pyramidal neurons in the rat piriform (olfactory) cortex from trained (TR) rats have reduced post-burst AHP for 3 days after odour-discrimination learning, and that this reduction is due to decreased conductance of calcium-dependent potassium current. In the present study, we examined whether this long-lasting reduction in AHP is mediated by second messenger systems. The broad-spectrum kinase inhibitor, H7, increased the AHP in neurons from TR rats, but not in neurons from pseudo-trained (pseudo-TR) and naive rats. Consequently, the difference in AHP amplitude between neurons from TR and control animals was diminished. This effect was also obtained by application of the specific PKC inhibitor, GF-109203x. The PKC activator, 1-Oleoyl-2-acetyl-sn-glycerol (OAG), significantly reduced the AHP in neurons from naive and pseudo-TR rats, but not in neurons from TR rats, so that the difference between the groups was abolished. The PKA-specific inhibitor, H-89, increased the AHP in neurons from all groups to a similar extent, and the difference in AHP amplitude between neurons from TR rats and neurons from controls was maintained. We suggest that while the post-burst AHP in piriform cortex pyramidal neurons is modulated by both PKC and PKA, a PKC-dependent process maintains the learning-related reduction of the AHP in these cells.

Learning-induced enhancement of postsynaptic potentials in pyramidal neurons.

We studied the effect of olfactory learning-induced modifications in piriform (olfactory) cortex pyramidal neurons on the propagation of postsynaptic potentials (PSPs). Rats were trained to distinguish between odors in pairs, in an olfactory discrimination task. Three days after training completion, PSPs were evoked in layer II pyramidal cells in piriform cortex brain slices by electrical stimulation of two pathways. Stimulation of layer Ib activated the intra-cortical fibers that terminate on the proximal region of the apical and basal dendrites. Stimulation of layer Ia activated the afferent axons that originate from the olfactory bulb and terminate on the distal apical dendrites. We have previously shown that olfactory training is accompanied by enhanced synaptic transmission in the intrinsic pathway, but not in the afferent pathway at 3 days after training. Here we show that at this stage, in both pathways PSPs evoked in neurons from trained rats had significantly faster rise time measured at the soma compared with PSPs in neurons from pseudo-trained and naive rats. Activation of the slow afterhyperpolarization (AHP), which is generated by potassium channels probably located at the proximal region of both apical and basal dendrites, reduced the amplitude measured at the soma of the proximal intrinsic pathway PSPs more effectively than PSPs that were generated distally by the afferent fibers. Thus the amount of reduction by AHP was used as a measure for the relative distance of PSP-generating sites from the soma. In neurons from trained rats, despite the previously reported reduction in AHP amplitude, AHP conductance shunted the PSPs from both synaptic pathways more efficiently compared with neurons from the control rats. We suggest that in neurons from trained rats PSPs are electrotonicly closer to the soma.

Synaptic contacts of neurons of fascia dentata transplants with nonspecific targets in neocortex of recipients

We carried out an electron microscopy study of possible synaptic contacts of the neurons of intracortical transplants of the rat brain fascia dentata with targets in the recipient somatosensory cortex. The axons of fascia dentata granular cell and their synaptic terminals could be easily identified in the neocortex due to their distinct morphological features (mossy fibers), although the fascia dentate cells normally do not interact with the neocortex. Thin nonmyelenized mossy fibers were found in both an intermediate zone between the transplant and brain and in the adjacent brain. Their presynaptic buds, like in situ, had large size and formed characteristic terminal, intraterminal, and en passant multiple synaptic contacts and desmosome-like junctions. The aberrant nerve fibers used perykaryons, dendrites of varying diameter, and dendrite spikes of the somatosensory cortex pyramidal neurons as postsynaptic targets in the neocortex. In addition to vacant spaces that appeared in the brain as a result of transplantation, the ingrowing axons induced the formation of additional contact sites: deep invaginations of the plasmalemma of perykaryons, somatic spikes, terminal branchings of dendrites, and dendritic outgrowths of complex branched shape. These aberrant contacts were characterized by the presence of polyribosomes, endoplasmic reticulum cisternae, and mitochondria in the postsynaptic loci. Osmiophility and extension of desmosome-like junctions were also enhanced in such synapses. Thus, it was shown that mossy fibers ingrowing in the recipient neocortex were capable of forming cell-to-cell contacts with signs of functional synapses to atypical cell targets.

Electrophysiological properties of pyramidal neurons in the rat prefrontal cortex: an in vivo intracellular recording study.

In order to determine the electrophysiological properties of prefrontal cortex pyramidal neurons in vivo, intracellular recordings coupled with neurobiotin injection were performed in anesthetized rats. Three main classes of pyramidal cells were distinguished according to both their firing patterns in response to depolarizing current pulses and the characteristics of their action potentials: regular spiking (RS, n = 71); intrinsic (inactivating) bursting (IB, n = 8); and non-inactivating bursting (NIB, n = 26) cells. RS cells were further subdivided into slow-adapting and fast-adapting types, according to their firing frequency adaptation. IB and fast-adapting RS cells could exhibit different firing patterns depending on the intensity of the depolarizing current. In response to successive depolarizing pulses of a given intensity, NIB and some RS cells showed variations in their firing patterns, probably due to the impact of local synaptic activity. All the labeled neurons were pyramidal cells with an apical dendrite that formed a terminal tuft in layer I. As compared to RS cells, NIB cells had a smaller somatic size and their apical dendritic tuft was less extensive, while IB cells presented a larger somatic size, thicker dendrites and a wider extent of their basal and apical dendritic arborization. In conclusion, we found in the rat prefrontal cortex, in vivo, different electrophysiological classes of pyramidal cells whose output firing patterns depend on interactions between their intrinsic properties and the ongoing synaptic activity.

Effects of cannabinoids on prefrontal neuronal responses to ventral tegmental area stimulation.

Cannabinoids activate the firing of mesoprefrontocortical dopamine neurons and release dopamine in the prefrontal cortex. This study was undertaken with the aim of clarifying the interaction between cannabinoids and mesocortical system in the prefrontal cortex. The effect of Delta9-tetrahydrocannabinol (Delta9-THC) and the synthetic CB1 agonist WIN55,212-2 (WIN) was studied by extracellular single unit recordings, in chloral hydrate anaesthetised rats, on the spontaneous activity of pyramidal neurons and on the inhibition produced on these neurons by the electrical stimulation of the ventral tegmental area (VTA). Intravenously administered Delta9-THC and WIN (1.0 and 0.5 mg/kg, respectively), increased the firing rate of pyramidal neurons projecting to the VTA. VTA stimulation produced a phasic inhibition (167 +/- 6 ms) in 79% of prefrontal cortex pyramidal neurons. Delta9-THC and WIN reverted this inhibition in 73% and 100% of the neurons tested, respectively. The subsequent administration of the selective CB1 antagonist SR141716A (1 mg/kg) readily suppressed the effects of both cannabinoids and restored the inhibitory response to VTA stimulation. Moreover, when administered alone, SR141716A prolonged the inhibition in 55.6% of the neurons tested. The results indicate that stimulation of CB1 receptors by cannabinoids results in an enhanced excitability of prefrontal cortex pyramidal neurons as indexed by the suppression of the inhibitory effect of VTA stimulation and by the increase in firing rate of antidromically identified neurons projecting to the VTA. Furthermore, our results support the view that endogenous cannabinoids exert a negative control on dopamine activity in the prefrontal cortex. This study may be relevant in helping to understand the influence of cannabinoids on cognitive processes mediated by the prefrontal cortex.

Cellular correlates of olfactory learning in the rat piriform cortex.

This review describes research that combines cellular physiology with behavioral neuroscience, to study the cellular mechanisms underlying learning and memory in the mammalian brain. Rats were trained with an olfactory conditioning paradigm, in which they had to memorize odors in order to be rewarded with drinking water. Such training results in rule learning, which enables enhanced acquisition of odor memory. Training results in the following learning-related physiological modifications in intrinsic and synaptic properties in olfactory (piriform) cortex pyramidal neurons: 1. increased neuronal excitability, indicated by reduced afterhyperpolarization, and 2. increased synaptic transmission, indicated by reduced paired-pulse facilitation. These modifications are correlated to enhanced learning capability rather than to storage of memory for specific odors. In addition, using a different paradigm of odor-training, it is shown that NMDA and betra-adrenergic receptors are involved at different stages of long-term memory consolidation.

Cannabinoids modulate synaptic strength and plasticity at glutamatergic synapses of rat prefrontal cortex pyramidal neurons.

Cannabinoids receptors have been reported to modulate synaptic transmission in many structures of the CNS, but yet little is known about their role in the prefrontal cortex where type I cannabinoid receptor (CB-1) are expressed. In this study, we tested first the acute effects of selective agonists and antagonist of CB-1 on glutamatergic excitatory postsynaptic currents (EPSCs) in slices of rat prefrontal cortex (PFC). EPSCs were evoked in patch-clamped layer V pyramidal cells by stimulation of layer V afferents. Monosynaptic EPSCs were strongly depressed by bath application (1 microM) of the cannabinoid receptors agonists WIN55212-2 (-50.4 +/- 8.8%) and CP55940 (-42.4 +/- 10.9%). The CB-1 antagonist SR141716A reversed these effects. Unexpectedly, SR141716A alone produced a significant increase of glutamatergic synaptic transmission (+46.9 +/- 11.2%), which could be partly reversed by WIN55212-2. In the presence of strontium in the bath, the frequency but not the amplitude of asynchronous synaptic events evoked in layer V pyramidal cells by stimulating layer V afferents, was markedly decreased (-54.2 +/- 8%), indicating a presynaptic site of action of cannabinoids at these synapses. Tetanic stimulation (100 pulses at 100 Hz, 4 trains) induced in control condition, no changes (n = 7/18), long-term depression (LTD; n = 6/18), or long-term potentiation (LTP; n = 5/18) of monosynaptic EPSCs evoked by stimulation of layer V afferents. When tetanus was applied in the presence of WIN 55,212-2 or SR141716-A (1 microM) in the bath, the proportion of "nonplastic" cells were not significantly changed (n = 7/15 in both cases). For the plastic ones (n = 8 in both cases), WIN 55,212-2 strongly favored LTD (n = 7/8) at the apparent expense of LTP (n = 1/8), whereas the opposite effect was observed with SR141716-A (7/8 LTP; 1/8 LTD). These results demonstrate that cannabinoids influence glutamatergic synaptic transmission and plasticity in the PFC of rodent.

Natural variability in the number of dendritic segments: Model-based inferences about branching during neurite outgrowth

A study was made of the possible basis for naturally occurring variations in the number of segments in individual dendritic trees. Distributions of the number of terminal segments have been studied in dendrites from rat, cat, and frog motoneurons, basal dendrites from rat visual cortex pyramidal and non-pyramidal neurons, in rat cerebellar Purkinje cell dendritic trees, and in human hippocampal dentate granule cells. By means of a mathematical model for dendritic branching, it was shown that the variation in the number of dendritic segments can be accounted for by assuming that new branches during neurite outgrowth are formed randomly at terminal segments. The observed terminal segment number distributions could be closely approximated by additionally assuming that branching probabilities decline with increasing number of terminal segments in growing dendrites. The pyramidal neuron group differed significantly from the other neuron groups in such a way as to suggest that this decline is stronger than in the dendrites of other types of neurons. By using literature data on the mean number of terminal segments in rat cerebellar Purkinje cells, measured at different times during early development, an estimate could be obtained of the time-course of the branching probabilities. The branching probability of a terminal segment was found to be in the order of 0.002 per hour in the first 4 weeks postnatal with a 5-fold transient increase in the second week.

Interactions between Associative Synaptic Potentials in the Piriform Cortex of the In Vitro Isolated Guinea Pig Brain

The interaction between synaptic potentials generated by the activation of separate sets of associative fibres was investigated in the piriform cortex of an in vitro isolated guinea pig brain preparation. Restricted regions of the piriform cortex served by separate contingents of afferent fibres of the lateral olfactory tract were isolated surgically. The activity generated by these patches of cortex in response to afferent stimulation propagates to remote cortical regions along cortico-cortical associative fibres. Current source density (CSD) analysis of field potential laminar profiles evoked by lateral olfactory tract stimulation confirmed that the synaptic sinks induced by distinct associative fibre contingents converge on the apical dendrites of piriform cortex neurons in the superficial lb layer. Pairing between potentials evoked by activation of two separate sets of associative fibres resulted in an almost linear summation when the two responses coincided. For interstimulus intervals of <100 ms, heterosynaptic pairing of independent associative inputs induced a facilitation of the conditioned associative potential, which correlated with an increase in the associative sink located in layer lb, as demonstrated by CSD analysis. The evaluation of the pairing intervals suggests that the heterosynaptic facilitation of the conditioned associative potentials may be due to the summation of local and remote associative synaptic events. It is concluded that separate associative inputs converge on the apical dendrites of piriform cortex pyramidal neurons to generate synaptic potentials through the activation of spatially close but independent synapses. The role of associative synaptic integration in the functional organization of the olfactory cortex is discussed.

Simulations of Intrinsically Bursting Neocortical Pyramidal Neurons

Neocortical layer 5 intrinsically bursting (IB) pyramidal neurons were simulated using compartment model methods. Morphological data as well as target neurophysiological responses were taken from a series of published studies on the same set of rat visual cortex pyramidal neurons (Mason, A. and Larkman, A. J., 1990. J. Neurosci. 9,1440-1447; Larkman, A. J. 1991. J. Comp. Neurol. 306, 307-319). A dendritic distribution of ion channels was found that reproduced the range of in vitro responses of layer 5 IB pyramidal neurons, including the transition from repetitive bursting to the burst/tonic spiking mode seen in these neurons as input magnitude increases. In light of available data, the simulation results suggest that in these neurons bursts are driven by an inward flow of current during a high threshold Ca2+ spike extending throughout both the basal and apical dendritic branches.

Synaptic and neuritic alterations during the progression of Alzheimer's disease.

Extensive synaptic and neuritic alterations in the neocortex and limbic system are characteristically found in Alzheimer's disease (AD). However, it is not known how early in the development of the disease these alterations occur. For the present study, we compared the synaptic and neuritic alterations among cases classified clinically and neuropathologically as early, mild and advanced AD. In early AD there was a 20% loss of synaptophysin-immunoreactive presynaptic terminals in the outer molecular layer of the hippocampal dentate gyrus (but not in the neocortex and entorhinal cortex), accompanied by increased amyloid precursor protein (APP) and Alz50 immunoreactivity in hippocampal and entorhinal cortex pyramidal neurons. These results suggest that abnormal neuronal expression of APP and cytoskeletal proteins in early stages might be involved in the mechanisms of synaptic pathology in AD.

Developmental regulation of Fos and Fos‐related antigens in cerebral cortex, striatum, hippocampus, and cerebellum of the rat

Previous work has associated the proto-oncogene c-fos with such events as neuronal excitation and cell growth and differentiation. This study specifically examined the expression of the Fos protein as well as other Fos-related antigens (Fras) during postnatal development of rat brain. Ages P1 through P15 as well as adult animals (P60) were examined. Particular focus was placed on developing cerebral cortex, striatum, hippocampus, and cerebellum. We used both the Alu antiserum, which recognizes the Fos protein specifically, and the M5 antiserum, which recognizes both Fos and a family of Fos-related antigens. Fos and Fras were developmentally regulated in a region- and cell-specific manner. Differential nuclear and cytoplasmic labeling appeared age dependent. Transient Fos expression was generally followed by a more protracted time course of Fra expression. Fos and a delayed or an extended expression of Fras were observed in subplate neurons between P1 and P15, in striatal striosome and matrix neurons between P1 and P9, and in hippocampal pyramidal neurons between P1 and P9. Fras alone were expressed in cerebral cortex pyramidal neurons and other cortical neurons between ages P1 and P15. Fos and Fras were concomitantly expressed in piriform and entorhinal cortical neurons between P1 and P9 and in cerebellar Purkinje cells between ages P5 and P10. Constitutive levels of Fos and Fras remained detectable in adult animals in a subset of cerebral cortical neurons and cerebellar Purkinje neurons.

Lead Blocks LTP by an Action Not at NMDA Receptors

Exposure of children to low levels of lead results in a reduction in cognitive ability and a series of behavioral deficits. We have studied the effects of PbCl 2 on long-term potentiation (LTP), the best available electrophysiologic model of learning and memory, in a rat piriform cortex brain slice preparation in order to test the hypothesis that lead neurotoxicity is a result of actions on LTP. With changes in the composition of the Krebs-Ringer solution normally used in brain slices, it is possible to keep the Pb 2+ in solution at concentrations up to 10 μ M. In this concentration range, Pb 2+ has no effect on the synaptic response elicited in piriform cortex pyramidal neurons upon stimulation of the lateral olfactory tract. We find that Pb 2+ blocks LTP by about 75% at 5 μ M and completely at 10 μ M. At these concentrations, Pb 2+ has no effect on posttetanic potentiation. Since it has been reported that Pb2+ blocks N-methyl- d-aspartate (NMDA) responses, and NMDA blockade is known at many sites to block LTP, we studied the effects of Pb 2+ on NMDA responses. In the concentration range studied there was no effect of Pb 2+ on NMDA responses. The mechanism whereby Pb 2+ blocks LTP remains to be determined. While the concentration of Pb 2+ found to block LTP in these studies is high relative to concentrations of Pb 2+ in blood that are associated with causing cognitive and behavioral effects in children, the sensitivity to Pb 2+ may be greater in young animals.

Immunohistochemistry of cholinergic receptors.

Acetylcholine and its receptors are involved in a variety of important signal transduction processes. As shown here paradigmatically for the human neuromuscular junction and the cerebral cortex, acetylcholine receptors can be visualized immunohistochemically at the cellular and subcellular level under physiological and pathological conditions. At normal motor endplates nicotinic cholinoceptors are localized at the surface of the postsynaptic junctional folds. In myasthenic syndromes investigation of muscle biopsies enables the diagnosis of receptor deficiencies at the ultrastructural level. In normal cerebral cortex pyramidal neurons are equipped with both nicotinic and muscarinic acetylcholine receptors localized to postsynaptic densities. In neuropsychiatric diseases cholinoceptor expression can be monitored at the cellular level by quantitative assessment of immunolabeled cortical neurons.

Net dendritic stability of layer II pyramidal neurons in F344 rat entorhinal cortex from 12 to 37 months

Dendritic extent of Golgi-Cox stained layer II entorhinal cortex pyramidal neurons was quantified in five groups of male F344 rats aged 12, 20, 27, 30 and 37 months. Over the age range studied, neither the apical nor the basal dendritic trees showed any statistically significant change in total dendritic length, numbers of segments or average segment length. This finding of average stability of the dendritic tree does not imply absence of remodelling of connections, but does require that if remodeling does occur, retraction and proliferation of dendrites must, on average, be equal. We hypothesized that in groups of animals with similar genetic and environmental histories neighbor neuron death provides the major stimulus for dendritic proliferation. Since we found dendritic stability in the cells reported here, we would predict that there should be no age-related loss of layer II pyramidal neurons in the entorhinal cortex of the normally aging F344 male rat between 12 and 37 months. This hypothesis may be tested by counting neurons within this region.

Differential effects of baclofen and gamma-aminobutyric acid (GABA) on rat piriform cortex pyramidal neurons in vitro.

1. The effects of baclofen and GABA on rat piriform cortex neurons were investigated electrophysiologically using a brain slice preparation. 2. At resting potential GABA depolarized and baclofen hyperpolarized the cell, probably through activation of Cl and K conductances acting at GABAA and GABAB receptors, respectively. 3. The GABAA receptors were concentrated on the apical and basal dendrites near the cell body, while the baclofen-sensitive GABA receptors were concentrated particularly on the basal dendrites. 4. The different distributions of receptor localization must have functional consequences which remain to be elucidated.

Effects of preweaning environmental enrichment on basilar dendrites of pyramidal neurons in occipital cortex: a Golgi study

The effects of postnatal environmental stimulation on the branching patterns of cortical dendrites were measured in rats. Pups were exposed to 4 daily multisensory enrichment sessions from days 10–24, while littermates were maintained in standard conditions. At 25 days of age, the brains were stained using the Golgi-Cox-Sholl method. Camera lucida drawings were made of the basilar dendritic trees from a total of 528 layer-III occipital cortex pyramidal neurons. A highly significant increase was found in number and length of segments from order 1–5 in the neurons from the enriched subjects.