Co-localization of vasoactive intestinal polypeptide, γ-aminobutyric acid and choline acetyltransferase in neocortical interneurons of the adult rat

Interneurons immunoreactive for vasoactive intestinal polypeptide (VIP) are integral elements of columnar organization patterns in the rat cerebral cortex. By application of the sensitive mirror technique, the co-localization of VIP with the classical inhibitory neurotransmitter γ-aminobutyric acid (GABA) and the acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT), was investigated in neocortical neurons. Furthermore, the frequency of co-localization of ChAT with GABA was determined. In a sample of 118 VIP-immunoreactive neurons, mostly from the primary somatosensory cortex, it was demonstrated that virtually all of them reveal immunoreactivity for GABA and, therefore, are to be GABAergic. Moreover, 34% of mostly bipolar, VIP-positive neurons contained ChAT and are, thus, supposedly cholinergic as well. Co-localization of VIP and ChAT varied according to cortical laminae. Finally, 88% of a total of 60 ChAT-immunoreactive neurons were also immunostained for GABA. It is concluded that almost all VIP-immunoreactive neurons and most of the cholinergic neurons in rat neocortex represent partly overlapping subpopulations of inhibitory interneurons utilizing GABA.     

Local circuit neurons of macaque monkey striate cortex: I. Neurons of laminae 4C and 5A

A study has been made, using Golgi preparations, of the organization of neurons with smooth or sparsely spined dendrites, here called local circuit neurons, of the macaque monkey primary visual cortex. Since these neurons include those responsible for inhibitory circuitry of the cortex, a better understanding of their anatomical organization is essential to concepts of functional organization of the region. This account describes those neurons found with cell body and major dendritic spread within the thalamic recipient zone of lamina 4C and its border zone with lamina 5A. The neurons are grouped firstly in terms of in which laminar division the soma occurred--4C beta, 4C alpha or the border zone of 5A-4C beta--and secondly, into varieties on the basis of the interlaminar projection patterns of their axons. Most, if not all, of the local circuit neurons of these divisions have interlaminar axon projections as well as an arbor local to their cell body and dendritic field. These interlaminar projections are highly specific, targeting from one to five laminar divisions depending on the variety of neuron; on this basis 17 varieties of local circuit neuron are described. While the number of varieties appears dauntingly large in terms of understanding the functional circuitry of the region, the clear-cut organization of the interlaminar links may provide clues as to the information processing that concerns each neuron. The local circuit neuron axon projections can be related to a wealth of information already available concerning the laminar organization of afferent axons and efferent cell groups, the organization of spiny neuron intrinsic relays (presumed to be excitatory), and physiological properties of different laminar divisions. It is hoped that the information derived from this study can serve as a guide for correlated physiological-anatomical studies on single cells of the region.     

Neural dynamics in cortex-striatum co-cultures-II. Spatiotemporal characteristics of neuronal activity

Neural dynamics in organotypic cortex-striatum co-cultures grown for three to six weeks under conditions of dopamine deficiency are described. Single neuron activities were recorded intra- and extracellularly, and spatiotemporal spreading of population activity was mapped using voltage-sensitive dyes. The temporal properties of spike firing were characterized by interspike interval histograms, autocorrelation and crosscorrelation.

Cortical pyramidal neurons (n = 40) showed irregular firing with a weak tendency to burst or to oscillate. Crosscorrelations revealed strong near-coincident firing and synaptic interactions. Disinhibition was a notable feature in a strongly firing cortical interneuron. Cortical activity spread in the co-culture, thus inducing an overall, homogeneous depolarization in the striatal part. Striatal cells were divided into principal cells and type I and II secondary cells. Principal cells (n = 40) were similar to those reported previously in vivo. Spiking activity ranged from irregular spiking at very low rates to episodic bursting, with an average burst duration of 1 s. Interspike intervals were single-peaked. Intracellular recordings revealed characteristic, long-lasting subthreshold depolarizations (“enabled state”) that were shortened by local muscarinic receptor blockade. During prolonged time periods in the “enabled state”, locally applied bicuculline induced strong firing in most principal neurons. Striatal secondary type I neurons (n = 25) showed high spiking rates, single- and double-peaked interval histograms and low-threshold, short-lasting stereotyped bursting activity and occasional rhythmic bursting. The firing of these neurons was increased by bicuculline. Crosscorrelations showed synchronization of these cells with principal cell activity. Secondary type II neurons (n = 15) revealed tonic, irregular firing patterns similar to cortical neurons, except with occasional firing in doublet spikes.

We conclude that under conditions of dopamine deficiency in corticostriatal co-cultures (i) the cortex induces the “enabled” state and typical bursting mode in striatal principal neurons; (ii) principal neurons are strongly inhibited during the “enabled” state; (iii) muscarinic activity, presumably from tonically active striatal cholinergic interneurons, stabilizes the “enabled” state; (iv) striatal GABAergic interneurons receive synaptic inhibition and take part in synchronized activity among striatal principal cells. Our results favor the view of the striatum as a lateral inhibition network.     

Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations

Using a new antibody developed against the C-terminus of the cannabinoid receptor (CB1), the immunostaining in the hippocampus revealed additional axon terminals relative to the pattern reported previously with an N-terminus antibody. Due to a greater sensitivity of this antibody, a large proportion of boutons in the dendritic layers displaying symmetrical (GABAergic) synapses were also strongly immunoreactive for CB1 receptors, as were axon terminals of perisomatic inhibitory cells containing cholecystokinin. Asymmetrical (glutamatergic) synapses, however, were always negative for CB1. To investigate the effect of presynaptic CB1 receptor activation on hippocampal inhibition, we recorded inhibitory postsynaptic currents (IPSCs) from principal cells. Bath application of CB1 receptor agonists (WIN55,212-2 and CP55,940) suppressed IPSCs evoked by local electrical stimulation, which could be prevented or reversed by the CB1 receptor antagonist SR141716A. Action potential-driven IPSCs, evoked by pharmacological stimulation of a subset of interneurons, were also decreased by CB1 receptor activation. We also examined the effects of CB1 receptor agonists on Ca2+-independent miniature IPSCs (mIPSC). Both agonists were without significant effect on the frequency or amplitude of mIPSCs. Synchronous gamma oscillations induced by kainic acid in the CA3 region of hippocampal slices were reversibly reduced in amplitude by the CB1 receptor agonist CP 55,940, which is consistent with an action on IPSCs. We used CB1-/- knock-out mice to confirm the specificity of the antibody and of the agonist (WIN55,212-2) action. We conclude that activation of presynaptic CB1 receptors decreases Ca2+-dependent GABA release, and thereby reduces the power of hippocampal network oscillations.     

Neuregulin and dopamine modulation of hippocampal gamma oscillations is dependent on dopamine D4 receptors

The neuregulin/ErbB signaling network is genetically associated with schizophrenia and modulates hippocampal γ oscillations-a type of neuronal network activity important for higher brain processes and altered in psychiatric disorders. Because neuregulin-1 (NRG-1) dramatically increases extracellular dopamine levels in the hippocampus, we investigated the relationship between NRG/ErbB and dopamine signaling in hippocampal γ oscillations. Using agonists for different D1- and D2-type dopamine receptors, we found that the D4 receptor (D4R) agonist PD168077, but not D1/D5 and D2/D3 agonists, increases γ oscillation power, and its effect is blocked by the highly specific D4R antagonist L-745,870. Using double in situ hybridization and immunofluorescence histochemistry, we show that hippocampal D4R mRNA and protein are more highly expressed in GAD67-positive GABAergic interneurons, many of which express the NRG-1 receptor ErbB4. Importantly, D4 and ErbB4 receptors are coexpressed in parvalbumin-positive basket cells that are critical for γ oscillations. Last, we report that D4R activation is essential for the effects of NRG-1 on network activity because L-745,870 and the atypical antipsychotic clozapine dramatically reduce the NRG-1-induced increase in γ oscillation power. This unique link between D4R and ErbB4 signaling on γ oscillation power, and their coexpression in parvalbumin-expressing interneurons, suggests a cellular mechanism that may be compromised in different psychiatric disorders affecting cognitive control. These findings are important given the association of a DRD4 polymorphism with alterations in attention, working memory, and γ oscillations, and suggest potential benefits of D4R modulators for targeting cognitive deficits.     

Cytoskeletal changes during development and aging in the cortex of neurofilament light protein knockout mice

The neurofilament light (NFL) subunit is considered as an obligate subunit polymer for neuronal intermediate filaments comprising the neurofilament (NF) triplet proteins. We examined cytoskeletal protein levels in the cerebral cortex of NFL knockout (KO) mice at postnatal day 4 (P4), 5 months, and 12 months of age compared with age‐matched wild‐type (WT) mice of a similar genetic background (C57BL/6). The absence of NFL protein resulted in a significant reduction of phosphorylated and dephosphorylated NFs (NF‐P, NF‐DP), the medium NF subunit (NFM), and the intermediate filament α‐internexin (INT) at P4. At 5 months, NF‐DP, NFM, and INT remained significantly lower in knockouts. At 12 months, NF‐P was again significantly decreased, and INT significantly increased, in KOs compared with wild type. In addition, protein levels of class III neuron‐specific β‐tubulin and microtubule‐associated protein 2 were significantly increased in NFL KO mice at P4, 5 months, and 12 months, whereas β‐actin levels were significantly decreased at P4. Immunocytochemical studies demonstrated that NF‐DP accumulated abnormally in the perikarya of cortical neurons by 5 months of age in NFL KO mice. Neurons that lacked NF triplet proteins, such as calretinin‐immunolabeled nonpyramidal cells, showed no alterations in density or cytoarchitectural distribution in NFL KO mice at 5 months relative to WT mice, although calretinin protein levels were decreased significantly after 12 months in NFL KO mice. These findings suggest that a lack of NFL protein alters the expression of cytoskeletal proteins and disrupts other NF subunits, causing intracellular aggregation but not gross structural changes in cortical neurons or cytoarchitecture. The data also indicate that changes in expression of other cytoskeletal proteins may compensate for decreased NFs. J. Comp. Neurol. 521:1817–1827, 2013. © 2012 Wiley Periodicals, Inc.     

Hilar interneuron vulnerability distinguishes aged rats with memory impairment

Hippocampal interneuron populations are reportedly vulnerable to normal aging. The relationship between interneuron network integrity and age‐related memory impairment, however, has not been tested directly. That question was addressed in the present study using a well‐characterized model in which outbred, aged, male Long‐Evans rats exhibit a spectrum of individual differences in hippocampal‐dependent memory. Selected interneuron populations in the hippocampus were visualized for stereological quantification with a panel of immunocytochemical markers, including glutamic acid decarboxylase‐67 (GAD67), somatostatin, and neuropeptide Y. The overall pattern of results was that, although the numbers of GAD67‐ and somatostatin‐positive interneurons declined with age across multiple fields of the hippocampus, alterations specifically related to the cognitive outcome of aging were observed exclusively in the hilus of the dentate gyrus. Because the total number of NeuN‐immunoreactive hilar neurons was unaffected, the decline observed with other markers likely reflects a loss of target protein rather than neuron death. In support of that interpretation, treatment with the atypical antiepileptic levetiracetam at a low dose shown previously to improve behavioral performance fully restored hilar SOM expression in aged, memory‐impaired rats. Age‐related decreases in GAD67‐ and somatostatin‐immunoreactive neuron number beyond the hilus were regionally selective and spared the CA1 field of the hippocampus entirely. Together these findings confirm the vulnerability of hippocampal interneurons to normal aging and highlight that the integrity of a specific subpopulation in the hilus is coupled with age‐related memory impairment. J. Comp. Neurol. 521:3508‐3523, 2013. © 2013 Wiley Periodicals, Inc.