Bacteriophage P4 DNA replication

The suprachiasmatic nucleus (SCN) is the circadian pacemaker in mammals and contains a network of arginine-vasopressin-immunoreactive (AVP-ir) neurons. AVP-recipient cells contain the V1a class of receptors linked to phosphoinositol turnover and protein kinase C (PKC). The present study describes the localization of AVP and the four Ca(2+)-dependent PKC-isoforms in the mouse and rabbit SCN. An estimate of the numerical density of AVP-ir neurons at the rostral, medial, and caudal level of the SCN revealed that the mouse SCN contains more than twice the number of AVP-ir neurons than the rabbit SCN. Neurons immunostained for AVP or PKC dominated in the dorsomedial and ventrolateral aspects of the mouse SCN, while the central area of the SCN revealed only weakly stained neurons. The rabbit SCN was characterized by a more homogeneous distribution of AVP-ir and PKC-ir neurons. PKC alpha was the most abundantly expressed isozyme in both species, whereas the presence of the other isoforms differed (mouse: PKC alpha > PKC beta I > PKC beta II > PKC gamma; rabbit: PKC alpha > PKC beta II > or = PKC gamma > PKC beta I). Clear PKC gamma-positive neurons were only observed in the rabbit SCN, while the mouse SCN predominantly contained immunolabeled fiber tracts for this PKC isozyme. Astrocytes immunoreactive for each PKC isoform were frequently encountered in the rabbit SCN, but were absent in mice. Immunofluorescence double labeling showed that numerous AVP-recipient cells in the mouse SCN were immunopositive for PKC alpha, and that nearly all AVP-ir neurons express PKC alpha abundantly. These results substantiate the putative role for PKC alpha in vasopressinergic signal transduction in the SCN. The differential expression in degree and cell type of the Ca(2+)-dependent PKC-isoforms in the mouse and rabbit SCN may be related to the differences observed in circadian timekeeping between the two species.     

A possible role for cholinergic neurons of the basal forebrain and pontomesencephalon in consciousness

Excitation at widely dispersed loci in the cerebral cortex may represent a neural correlate of consciousness. Accordingly, each unique combination of excited neurons would determine the content of a conscious moment. This conceptualization would be strengthened if we could identify what orchestrates the various combinations of excited neurons. In the present paper, cholinergic afferents to the cerebral cortex are hypothesized to enhance activity at specific cortical circuits and determine the content of a conscious moment by activating certain combinations of postsynaptic sites in select cortical modules. It is proposed that these selections are enabled by learning-related restructuring that simultaneously adjusts the cytoskeletal matrix at specific constellations of postsynaptic sites giving all a similar geometry. The underlying mechanism of conscious awareness hypothetically involves cholinergic mediation of linkages between microtubules and microtubule-associated protein-2 (MAP-2). The first reason for proposing this mechanism is that previous studies indicate cognitive-related changes in MAP-2 occur in cholinoceptive cells within discrete cortical modules. These cortical modules are found throughout the cerebral cortex, measure 1-2 mm2, and contain approximately 10(3)-10(4) cholinoceptive cells that are enriched with MAP-2. The subsectors of the hippocampus may function similarly to cortical modules. The second reason for proposing the current mechanism is that the MAP-2 rich cells throughout the cerebral cortex correspond almost exactly with the cortical cells containing muscarinic receptors. Many of these cholinoceptive, MAP-2 rich cells are large pyramidal cell types, but some are also small pyramidal cells and nonpyramidal types. The third reason for proposing the current mechanism is that cholinergic afferents are module-specific; cholinergic axons terminate wholly within individual cortical modules. The cholinergic afferents may be unique in this regard. Finally, the tapering apical dendrites of pyramidal cells are proposed as primary sites for cholinergic mediation of linkages between MAP-2 and microtubules because especially high amounts of MAP-2 are found here. Also, the possibility is raised that muscarinic actions on MAP-2 could modulate microtubular coherence and self-collapse, phenomena that have been suggested to underlie consciousness.     

Transcriptional down-regulation of myogenin expression is associated with v-ras-induced block of differentiation in unestablished quail muscle cells

This study examined the distribution of muscarinic acetylcholine receptor-immunoreactive neurons in the amygdaloid complex of the rat, with emphasis on the central nucleus. The monoclonal antibody M35 raised against purified muscarinic acetylcholine receptor protein was used to visualize muscarinic acetylcholine receptor-immunoreactive cells. Muscarinic acetylcholine receptor immuno-reactivity was high in the central nucleus and low to moderate in all other regions of the amygdaloid complex. Within the central nucleus, the muscarinic acetylcholine receptor-immunoreactive neurons were found predominantly in the lateral subdivision. This region contained medium-sized neurons (largest diameter ranging from 10 to 15 microns), with a round or slightly ovoid cell shape. At the subcellular level, however, the labeled neurons revealed relatively few muscarinic acetylcholine receptor-immunoreactive postsynaptic densities. Immunofluorescent double-labeling demonstrated that nearly all of the muscarinic acetylcholine receptor-immunoreactive neurons (98.6%) in the central nucleus expressed abundant amounts of nicotinic acetylcholine receptors, further substantiating the cholinoceptive character of these cells. In addition, the vast majority of these muscarinic acetylcholine receptor-immunoreactive neurons (94.3%) were GABAergic neurons. The muscarinic acetylcholine receptor-immunoreactive neurons expressed moderate levels of protein kinase gamma, one of the likely intracellular mediators between muscarinic acetylcholine receptors and their elicited physiological response. The number and staining intensity of muscarinic acetylcholine receptor-immunoreactive neurons in the central nucleus varied dramatically among rats. This individual variation correlated positively with the rat's expression of conditioned immobility and correlated negatively with active shock avoidance performance. These results suggest that the GABAergic/cholinoceptive neuronal elements in the central nucleus are involved in the expression of fear-induced behaviors. This interpretation is further elaborated in a forthcoming paper.     

Characterization of neocortical non-pyramidal neurons expressing preprotachykinins A and B: a double immunofluorescence study in the rat

Neurons expressing preprotachykinin A and preprotachykinin B, which are the precursor prepropeptides of substance P and neurokinin B (neuromedin K), respectively, were characterized immunocytochemically in the rat neocortex. Antibodies raised against C-terminal portions of preprotachykinins were used for labeling cell bodies of preprotachykinin-producing neurons. Neurons immunoreactive for preprotachykinin B were encountered four times more frequently in the neocortex than those immunoreactive for preprotachykinin A. Preprotachykinin A-immunoreactive neurons were scattered more frequently in the deep cortical layers (layers IV-VI) than in the superficial layers (layers I-III), whereas preprotachykinin B-immunoreactive neurons were distributed more frequently in the superficial layers than in the deep layers. Almost all preprotachykinin-expressing neurons were immunoreactive for GABA, suggesting that they were non-pyramidal cells. However, co-expression of the two preprotachykinin immunoreactivities in single neurons was not found. Preprotachykinin-expressing neocortical neurons were further examined with markers for subpopulations of GABAergic cortical neurons. Immunoreactivities for parvalbumin, calbindin and somatostatin were found in 69%, 27% and 11%, respectively, of preprotachykinin A-immunoreactive neurons. Conversely, preprotachykinin A-immunoreactive neurons constituted only 6% of parvalbumin-immunoreactive neurons, 4% of calbindin-immunoreactive neurons and 1% of somatostatin-immunoreactive neurons. Immunoreactivities for calretinin, choline acetyltransferase, vasoactive intestinal polypeptide, corticotropin-releasing factor and cholecystokinin were detected in 13-39% of preprotachykinin B-immunoreactive neurons. Preprotachykinin B immunoreactivity was seen in 33% of calretinin-positive neurons, 45% of cholinergic neurons, 47% of vasoactive intestinal polypeptide-positive neurons, 59% of corticotropin-releasing factor-positive neurons and 83% of cholecystokinin-positive neurons. These results indicate that preprotachykinin A- and preprotachykinin B-expressing neurons constitute separate populations of GABAergic non-pyramidal neurons in the neocortex. Since receptors for substance P and neurokinin B are expressed in GABAergic neurons [Kaneko T. et al. (1994) Neuroscience 60, 199-211] and pyramidal neurons [Ding Y. Q. et al. (1996) J. comp. Neurol. 364, 290-310], respectively, cortical neurons may use two separate lines of tachykinin signals; substance P serves as a signal between GABAergic non-pyramidal neurons, whereas neurokinin B acts as a signal of GABAergic neurons to pyramidal neurons.     

Selective disruption by protein kinases of G-protein-mediated Ca2+ channel modulation

1. We studied the effects of phorbol-12-myristate, 13-acetate (PMA) on G-protein-mediated inhibition of Ca2+ channels by several neurotransmitters in rat superior cervical ganglion (SCG) sympathetic neurons, with the use of the whole cell patch clamp. PMA attenuated membrane-delimited inhibition of calcium currents (ICa) by norepinephrine (NE) and somatostatin by more than half, but did not attenuate inhibition by M1 muscarinic receptors, which use a diffusible cytoplasmic messenger. Inhibition of ICa by NE through pertussis-toxin-sensitive and -insensitive G proteins was equally attenuated by PMA. PMA enhanced ICa in about half the neurons (enhancement of 10 +/- 1%, mean +/- SE) and strongly reduced the holding current in 44 of 61 cells. 2. The M-type K+ current (IM) was not suppressed by PMA, and PMA did not attenuate inhibition of IM by muscarinic agonists, which is also via a diffusible cytoplasmic messenger. 3. Attenuation of NE and somatostatin inhibition by PMA was blocked by 1 microM staurosporine, a broad-spectrum protein kinase inhibitor. Tests with three inhibitors selective for distinct isoforms of protein kinase C (PKC) gave mixed results. PMA's actions were unaffected by 1 microM calphostin C, blocked by 500 nM bisindolylmaleimide, and unaffected by the pseudosubstrate inhibitor PKC19-36. 4. Thus we find that two membrane-delimited signaling pathways that inhibit ion channels in rat SCG neurons are strongly attenuated by PMA, but signaling pathway(s) that use a diffusible cytoplasmic messenger are not. We speculate that a nonstandard PKC isoform, perhaps PKC mu, mediates PMA actions.     

Muscarinic M1 receptor mediated inhibition of GABA release from rat cerebral cortex

Presynaptic modulation of [3H]GABA release was examined using rat cerebral cortical slices. In vitro addition of carbachol, a muscarinic receptor agonist, resulted in a significant suppression of the release of [3H]GABA evoked by high potassium (50 mM) stimulation in a dose dependent manner, while noradrenaline, isoproterenol, dopamine, 5-hydroxytryptamine, histamine and glutamic acid had no significant effect on the evoked release of [3H]GABA. This suppressive effect of carbachol was antagonized invariably by atropine. Furthermore, it was found that the suppressive action of carbachol could be antagonized by pirenzepine, a selective M1 muscarinic receptor antagonist, but not by AF-DX 116 and 4-DAMP, M2 and M3 receptor antagonists, respectively. These results suggest that the release of GABA from cerebral cortical GABA neurons may be modulated by presynaptic M1 muscarinic receptor.     

Loss of muscarinic receptors and of stimulated phospholipid labeling in ibotenate-treated hippocampus

The stimulation of phospholipid labeling by muscarinic agonists has been examined in nerve ending preparations from lesioned hippocampus in order to investigate the synaptic locus of the effect. Unilateral injections of the neurotoxin, ibotenic acid, into the hippocampus resulted in an extensive loss of nerve cells from both the dentate gyrus and hippocampus on the lesioned side and a parallel loss of muscarinic receptors as revealed by [3H]quinuclidinyl benzilate autoradiography. Homogenates and nerve ending fractions prepared from the lesioned side of the hippocampus possessed a reduced specific activity (expressed per milligram of protein) of glutamic acid decarboxylase as well as a reduced number of muscarinic receptors compared with the control side. By contrast, choline acetyltransferase activity was either unchanged or slightly increased on the lesioned side. Although there was a reduced yield (25%) of nerve endings from the lesioned side, the specific activity of 32Pi incorporation into phospholipids in the absence of added carbachol was comparable to that of the control side. There was, however, a marked reduction in the carbachol stimulation of phosphatidic acid and phosphatidylinositol labeling in nerve ending fractions obtained from he lesioned hippocampus. These results indicate that the muscarinic receptors present in nerve ending fractions from hippocampus and implicated in stimulated phospholipid turnover are derived from cholinoceptive intrinsic neurons.     

Distribution and translocation of isoforms of protein kinase C in rat submandibular acinar cells

The distribution of six isoforms of protein kinase C (PKC) in seromucous acinar cells of rat submandibular gland was examined and their translocation from the cytosolic- to the membrane fraction after different stimuli investigated. Western blotting, immunostaining with isoform-specific antibodies and scanning densitometry showed that PKC-alpha and epsilon were distributed fairly evenly between the cytosol and membranes in resting cells, while isoforms- beta, delta and zeta were all predominantly localized (over 80%) in membranes. PKC-gamma was not detected. PKC-alpha was mobilized to the membrane fraction by the phorbol ester, TPA, but not by the phosphoinositide-coupled agonists carbachol, methoxamine and substance P (SP). PKC-epsilon was translocated by TPA and carbachol but not by SP or methoxamine. Biochemical assay of total PKC confirmed that cytosolic enzyme activity was significantly reduced by TPA and carbachol to 29% and 75% respectively of control levels. These results suggest that muscarinic regulation of the mucosecretory response in the rat submandibular gland may be mediated by the PKC-epsilon isoform.     

Differential cellular distribution and dynamics of HSP70, cyclooxygenase-2, and c-Fos in the rat brain after transient focal ischemia or kainic acid

Cerebral ischemia and also excitotoxicity induce the expression of 72,000 mol. wt heat shock protein (Hsp70), c-Fos, and cyclooxygenase-2. In the present work we have examined whether Hsp70, c-Fos and cyclooxygenase-2 are expressed by the same cells in the rat brain at 6, 12 and 24 h following transient focal ischemia or kainic acid administration, by means of single and double immunohistochemistry. At 6 h after kainic acid, some co-localization of Hsp70 with c-Fos and cyclooxygenase-2 was seen in pyramidal hippocampal neurons and superficial cortical layers, however by 24 h such colocalization became rare within the cortex but was partially maintained in the hippocampus. Cyclooxygenase-2 was seen in many neurons that were also immunoreactive for c-Fos in superficial cortical layers, dentate gyrus and pyramidal cell layer of the hippocampus from 6 h after kainic acid. Co-localization of cyclooxygenase-2 and c-Fos was also observed in superficial cortical layers within the ipsilateral hemisphere at 6 h following focal ischemia. Also, some co-localization of Hsp70 with c-Fos and cyclooxygenase-2 was seen at this time. However, by 24 h cyclooxygenase-2 and c-Fos-immunoreactive cells were restricted to perifocal regions, and only a very limited co-localization with Hsp70 was seen in perifocal neurons located in the border of the penumbra-like area that surrounds the ischemic core and is strongly immunoreactive for Hsp70. This study shows a selective and dynamic cellular expression of inducible proteins following either ischemia or kainic acid, with a remarkable neuronal co-localization of c-Fos and cyclooxygenase-2. The results suggest that, first, stimuli underlying neuronal c-Fos expression can also lead to the induction of cyclooxygenase-2; second, transient co-localization of Hsp70 and c-Fos can take place in non-vulnerable neurons; and finally, expression of c-Fos, cyclooxygenase-2, and/or Hsp70 at a given time-point is part of the response to altered environmental conditions and can be related to the particular cellular sensitivity rather than the pathological outcome.     

Infracortical interstitial cells concurrently expressing m2-muscarinic receptors, acetylcholinesterase and nicotinamide adenine dinucleotide phosphate-diaphorase in the human and monkey cerebral cortex

Intense immunoreactivity for the m2-muscarinic receptor was found in a population of interstitial polymorphic neurons embedded within the infracortical white matter and the adjacent deep layers of the cerebral cortex. These infracortical neurons were evenly distributed throughout architectonic subdivisions of the monkey cortex except for parts of primary visual cortex where they were less numerous. A similar set of m2-immunoreactive interstitial cells was also detected in the human lateral temporal neocortex obtained at surgery. Upon electron microscopic examination, they were found to receive unlabelled synaptic inputs and displayed abundant rough endoplasmic reticulum, a prominent nucleolus, and invaginations of the nuclear membrane. Double labelling of m2 immunoreactivity and acetylcholinesterase histochemistry demonstrated that approximately 90% of the m2-positive infracortical cells were acetylcholinesterase-rich in the monkey and human brains. Conversely, the proportion of acetylcholinesterase-rich infracortical neurons that were m2-immunoreactive was over 90% in the monkey and at least 50% in the human. The concurrent visualization of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) enzyme activity with m2 immunoreactivity in the monkey and human brain showed that 85-95% of m2-immunoreactive infracortical cells were NADPH-d positive. Conversely, about 70% of NADPH-d cells contained m2 immunoreactivity. These observations provide the most convincing information to date that many of the acetylcholinesterase-rich neurons located in the infracortical white matter of the cerebral cortex are likely to be cholinoceptive. The expression of NADPH-d by these neurons suggests that they may also provide a relay through which cholinergic innervation, originating predominantly from the nucleus basalis of Meynert, could regulate the release of nitric oxide in the cerebral cortex and subjacent white matter. The degeneration of these neurons may account for at least some of the depletion of m2 receptors that has been reported in Alzheimer's disease.     

Carbachol stimulates c-fos expression and proliferation in oligodendrocyte progenitors

To determine if muscarinic receptor-activation plays a role in oligodendrocyte development, the effect of carbachol a stable acetylcholine analog, on gene expression and proliferation was investigated. Using Northern blot analysis we showed that carbachol caused a time and concentration-dependent increase in c-fos mRNA. This effect was blocked by atropine, a non-selective muscarinic antagonist. In addition, the muscarinic-stimulated c-fos increase was inhibited by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C (PKC), but not by N-2-(p-bromocinnamylamino)-ethyl-5-isoquinoline-sulfonamide (H-89), a potent inhibitor of protein kinase A, suggesting the involvement of PKC in mediating the response. Down-regulation of PKC by overnight pre-treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) blocked only the phorbol ester-stimulated c-fos accumulation while no effect was observed in the carbachol-induced response. These results suggested that carbachol stimulated an H-7 sensitive PKC pathway which may be different than that activated by TPA. Further evidence for two separate mechanisms of proto-oncogene induction was provided by the additive effect of carbachol and TPA. Induction of c-fos mRNA by carbachol was dependent on both influx of extracellular Ca2+ and release from intracellular stores, as both EDTA and BAPTA blocked the response. Since activation of muscarinic receptors can affect cell division in other cellular systems, the effect of carbachol on [3H]thymidine and bromodeoxyuridine incorporation into oligodendrocyte DNA was measured. Carbachol stimulated DNA synthesis in oligodendrocyte progenitors. This effect was mediated by muscarinic receptors as [3H]thymidine incorporation was prevented or significantly reduced by the addition of atropine. In conclusion, the present findings suggest that, the neurotransmitter, acetylcholine may act as a trophic factor in developing oligodendrocytes, regulating their growth and development in the central nervous system.     

Cell- and lamina-specific expression and activity-dependent regulation of type II calcium/calmodulin-dependent protein kinase isoforms in monkey visual cortex

In situ hybridization histochemistry and immunocytochemistry were used to study localization and activity-dependent regulation of alpha, beta, gamma, and delta isoforms of type II calcium/calmodulin-dependent protein kinase (CaMKII) and their mRNAs in areas 17 and 18 of normal and monocularly deprived adult macaques. CaMKII-alpha is expressed overall at levels three to four times higher than that of CaMKII-beta and at least 15 times higher than that of CaMKII-gamma and -delta. All isoforms are expressed primarily in pyramidal cells of both areas, especially those of layers II-III, IVA (in area 17), and VI, but are also expressed in nonpyramidal, non-GABAergic cells of layer IV of both areas and in interstitial neurons of the white matter. CaMKII-alpha and -beta are colocalized, suggesting the formation of heteromers. There was no evidence of expression in neuroglial cells. Each isoform has a unique pattern of laminar and sublaminar distribution, but cortical layers or sublayers enriched for one isoform do not correlate with layers receiving inputs only from isoform-specific layers of the lateral geniculate nucleus. CaMKII-alpha and -beta mRNA and protein levels in layer IVC of area 17 are subject to activity-dependent regulation, with brief periods of monocular deprivation caused by intraocular injections of tetrodotoxin leading to a 30% increase in CaMKII-alpha mRNA and a comparable decrease in CaMKII-beta mRNA in deprived ocular dominance columns, especially of layer IVCbeta. Expression in other layers and expression of CaMKII-gamma and delta were unaffected. Changes occurring in layer IVC may influence the formation of heteromers and protect supragranular layers from CaMKII-dependent plasticity in the adult.     

Current excitement from insect muscarinic receptors

Recent electrophysiological, pharmacological and molecular studies suggest that muscarinic ACh receptors (mAChRs) in insects are related to, but distinct from, their mammalian counterparts. Insect mAChRs perform two primary roles that are distinguished by their locations. Presynaptic mAChRs, present on sensory terminals, inhibit transmitter release, thereby reducing the effectiveness of specific afferent inputs. In contrast, postsynaptic mAChRs depolarize and increase the excitability of motoneurons and interneurons, thereby acting as dynamic-gain controls. This postsynaptic modulation is achieved in different ways in specific neurons but generally results from the activation of persistent inward and outward currents. At the level of neural processing, these distinct roles enable insect mAChRs to regulate the transfer of sensory information, and modulate the contributions of central neurons to central pattern generators and reflexes. Because these phenomena can be studied in identified neurons, a combination of physiological and molecular studies of mAChRs in insects should help to elucidate some of their behavioral roles. Furthermore, such studies could lead to the identification of general mechanisms of functional plasticity in neuronal networks.     

Differential cholinoceptor subtype-dependent activation of signal transduction pathways in neonatal versus adult rat atria

In this study, we investigated the expression and distribution of muscarinic cholinergic receptors (mAChRs) and the different signaling pathways associated with mAChR activation in atria isolated from adult and neonatal rats. Carbachol stimulation of mAChRs in both neonatal and adult rat atria led to a negative inotropic response with activation of phosphoinositide hydrolysis, an increase in cyclic GMP levels, and a decrease in cyclic AMP production. However, compared with adult atria, neonatal atria showed hypersensitivity in the contractile effect induced by carbachol. Pharmacological analysis with mAChR antagonists indicated that M1 and M2 mAChR subtypes are important mediators of the response to carbachol in neonatal atria. In contrast, in adult atria the effect of the agonist was coupled only to the M2 mAChR subtype. Moreover, an increased number of total mAChRs was labeled in neonatal atrial membranes compared with those of adults. Although a predominant M2 mAChR population is expressed in atria at both stages of development studied, competition binding parameters calculated for carbachol indicated the presence of high-affinity binding sites, with higher affinity in neonates than in adults. These results suggest that the differences observed between neonatal and adult atria in their response to a cholinergic agonist may be related to differential expression of mAChR subtypes and/or changes in functional coupling of mAChR subtypes during development.     

Isoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons

High concentrations of glutamate, the major excitatory neurotransmitter in the mammalian brain, lead to intracellular calcium overload resulting in excitotoxic damage and death of neurons. Since protein kinase C (PKC) is involved in neuronal degeneration resulting from cerebral ischemia and from glutamate excitotoxicity, we investigated the effect of glutamate on changes in the cellular distribution of various PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by the PKC activator phorbol ester. Out of the expressed PKC isoforms alpha, gamma, epsilon, zeta and lambda only the conventional isoforms PKC alpha and gamma responded to glutamate. Using subcellular fractionation and Western blotting with isoform-specific antibodies and immunocytochemical localization with confocal laser scanning microscopy, we observed that phorbol ester and glutamate have different effects on PKC isoform redistribution: Whereas phorbol ester resulted in translocation of PKC alpha and PKC gamma toward a membrane fraction, the glutamate-mediated rise in intracellular calcium concentration induced a translocation mainly toward a detergent-insoluble, cytoskeletal fraction. Immunocytochemical analysis revealed an isoform-specific translocation following glutamate treatment: PKC gamma was translocated mainly to cytoplasmic, organelle-like structures, whereas PKC alpha redistributed to the plasma membrane and into the cell nucleus. The latter result is of special interest, as it indicates that nuclear PKC may play a role in processes of excitotoxic cell damage.     

Differential effects of acetylcholine on neuronal activity and interactions in the auditory cortex of the guinea-pig

During normal brain operations, cortical neurons are subjected to continuous cholinergic modulations. In vitro studies have indicated that, in addition to affecting general cellular excitability, acetylcholine also modulates synaptic transmission. Whether these cholinergic mechanisms lead to a modulation of functional connectivity in vivo is not yet known. Herein, the effects were studied of an iontophoretic application of acetylcholine and of the muscarinic agonist, carbachol, on the ongoing activity and co-activity of neurons simultaneously recorded in the auditory cortex of the anaesthetized guinea-pig. Iontophoresis of cholinergic agonists mainly affected the spontaneous firing rates of auditory neurons, affected autocorrelations less (in most cases their central peak areas were reduced), and rarely affected cross-correlations. These findings are consistent with cholinergic agonists primarily affecting the excitability of cortical neurons rather than the strength of cortical connections. However, when changes of cross-correlations occurred, they were usually not correlated with concomitant changes in average firing rates nor with changes in autocorrelations, which suggests a secondary cholinergic effect on specific cortico-cortical or thalamo-cortical connections.     

Dopamine innervation of a subclass of local circuit neurons in monkey prefrontal cortex: ultrastructural analysis of tyrosine hydroxylase and parvalbumin immunoreactive structures

The ability of dopamine to regulate the cognitive functions of the prefrontal cortex (PFC) involves complex modulatory actions on GABA-containing local circuit neurons in addition to pyramidal cells. However, the subclasses of cortical neurons that receive direct dopamine input are not known. We sought to determine whether dopamine terminals innervate the subclasses of local circuit neurons that contain the calcium-binding protein parvalbumin (PV), namely the wide arbor and chandelier neurons that target pyramidal cell soma and axon initial segments respectively. Sections through area 9 of five monkeys were labeled with immunoperoxidase for tyrosine hydroxylase (TH), to identify dopamine terminals, and with immunogold-silver for PV. Electron microscopic examination of the middle cortical layers (IIIb-IV) revealed that TH-positive terminals were sometimes directly apposed to PV-labeled dendrites, and approximately one-third of these contacts exhibited morphological features that are typically associated with symmetric synapses. In contrast, TH-immunolabeled terminals in the superficial layers (I-IIIa) were less frequently apposed to PV-positive dendrites, and none of these contacts exhibited synapse-like morphology. These findings, in concert with previous studies of GABA- or calretinin-containing local circuit neurons, suggest that dopamine's modulatory action in the PFC involves selective effects on only certain interneuron populations, including those that mediate potent inhibitory actions on pyramidal cells.     

Increases in protein kinase C gamma immunoreactivity in the spinal cord of rats associated with tolerance to the analgesic effects of morphine

Our previous studies have indicated a critical role of protein kinase C (PKC) in intracellular mechanisms of tolerance to morphine analgesia. In the present experiments, we examined (1) the cellular distribution of a PKC isoform (PKC gamma) in the spinal cord dorsal horn of rats associated with morphine tolerance by utilizing an immunocytochemical method and (2) the effects of the N-methyl-D-aspartate receptor antagonist MK-801 on tolerance-associated PKC gamma changes. In association with the development of tolerance to morphine analgesia induced by once daily intrathecal administration of 10 micrograms morphine for eight days, PKC gamma immunoreactivity was clearly increased in the spinal cord dorsal horn of these same rats. Within the spinal cord dorsal horn of morphine tolerant rats, there were significantly more PKC gamma immunostained neurons in laminae I-II than in laminae III-IV and V-VI. Such PKC gamma immunostaining was observed primarily in neuronal somata indicating a postsynaptic site of PKC gamma increases. Moreover, both the development of morphine tolerance and the increase in PKC gamma immunoreactivity were prevented by co-administration of morphine with 10 nmol MK-801 between Day 2 and Day 7 of the eight day treatment schedule. In contrast, PKC gamma immunoreactivity was not increased in rats receiving a single i.t. administration of 10 micrograms morphine on Day 8, nor did repeated treatment with 10 nmol MK-801 alone change baseline levels of PKC gamma immunoreactivity. These results provide further evidence for the involvement of PKC in NMDA receptor-mediated mechanisms of morphine tolerance.     

Induction of c-fos expression in hypothalamic magnocellular neurons requires synaptic activation and not simply increased spike activity

Magnocellular neurons of the hypothalamic supraoptic nucleus have been shown to express the immediate-early gene c-fos in a number of experimental and physiological circumstances. In each case the induction of the immediate-early gene followed the increase in the spike activity of the cells. Since an increase in the intracellular concentration of calcium following influx through voltage-sensitive calcium channels is a known stimulus for c-fos expression and since the action potentials of these neurons have a large calcium component, we hypothesized that c-fos induction in these neurons could be attributed to calcium influx during spike activity. In the present experiments we use extracellular recording and immunocytochemistry for Fos, the protein product of c-fos, to demonstrate the activation of the cells following intracerebroventricular administration of the muscarinic agonist, carbachol. Fos expression following carbachol injection was then compared with that induced by a similar number of antidromically evoked action potentials. Antidromic activation, unlike the activation induced by carbachol, did not lead to the induction of Fos. We conclude that Fos induction in these neurons requires receptor activation rather than spike activity.