Discrepancy between antennal and behavioral responses for enantiomers of alpha-pinene: electrophysiology and behavior of Helicoverpa armigera (Lepidoptera).

The ability of adult cotton bollworm, Helicoverpa armigera (Hübner), to distinguish and respond to enantiomers of alpha-pinene was investigated with electrophysiological and behavioral methods. Electroantennogram recordings using mixtures of the enantiomers at saturating dose levels, and single unit electrophysiology, indicated that the two forms were detected by the same receptor neurons. The relative size of the electroantennogram response was higher for the (-) compared to the (+) form, indicating greater affinity for the (-) form at the level of the dendrites. Behavioral assays investigated the ability of moths to discriminate between, and respond to the (+) and (-) forms of alpha-pinene. Moths with no odor conditioning showed an innate preference for (+)-alpha-pinene. This preference displayed by naive moths was not significantly different from the preferences of moths conditioned on (+)-alpha-pinene. However, we found a significant difference in preference between moths conditioned on the (-) enantiomer compared to naive moths and moths conditioned on (+)-alpha-pinene, showing that learning plays an important role in the behavioral response. Moths are less able to distinguish between enantiomers of alpha-pinene than different odors (e.g., phenylacetaldehyde versus (-)-alpha-pinene) in learning experiments. The relevance of receptor discrimination of enantiomers and learning ability of the moths in host plant choice is discussed.

Olfaction in the Queensland fruit fly, Bactrocera tryoni. I: Identification of olfactory receptor neuron types responding to environmental odors.

The electroantennogram method was used to investigate the number of distinct olfactory receptor neuron types responding to a range of behaviorally active volatile chemicals in gravid Queensland fruit flies, Bactrocera tryoni. Three receptor neuron types were identified. One type responds to methyl butyrate, 2-butanone, farnesene, and carbon dioxide; a second to ethanol; and a third to n-butyric acid and ammonia. The receptor neuron type responding to methyl butyrate, 2-butanone, farnesene, and carbon dioxide consists of three subtypes. The presence of a limited number of receptor neuron types responding to a diverse set of chemicals and the reception of carbon dioxide by a receptor neuron type that responds to other odorants are novel aspects of the peripheral olfactory discrimination process.

Olfaction in the Queensland fruit fly, Bactrocera tryoni. II: Response spectra and temporal encoding characteristics of the carbon dioxide receptors.

Single-unit electrophysiology was used to record the nerve impulses from the carbon dioxide receptors of female Queensland fruit flies, Bactroera tryoni. The receptors responded to stimulation in a phasic-tonic manner and also had a period of inhibition of the nerve impulses after the end of stimulation. at high stimulus intensities. The cell responding to carbon dioxide was presented with a range of environmental odorants and found to respond to methyl butyrate and 2-butanone. The coding characteristics of the carbon dioxide cell and the ability to detect other odorants are discussed, with particular reference to the known behavior of the fly.

Altered excitatory amino acid function and morphology of the cerebellum of the spastic Han-Wistar rat.

A mutant strain of Han-Wistar rat carries an autosomal recessive gene producing spastic paresis which is characterized by ataxia, tremor and hind limb rigidity. Brains of affected rats and unaffected littermate controls were transected at the mesencephalon into rostral and caudal portions (the caudal portion contained the cerebellum and brainstem). Poly(A)+ mRNA was isolated from pooled rostral or caudal portions and injected into Xenopus oocytes. The oocytes were voltage-clamped and exposed to 1 mM L-glutamate, 500 microM kainate, 500 microM quisqualate, 200 microM N-methyl-D-aspartate (NMDA) or 1 mM gamma-aminobutyric acid (GABA). Oocytes injected with mRNA isolated from the caudal portions of the affected rat brains exhibited statistically significant increases in glutamate and kainate peak current responses compared to oocytes injected with mRNA from other brain samples. No differences were noted in the responses of the groups when exposed to quisqualate, NMDA or GABA. Cerebellar and brain stem mRNA were also isolated separately in different groups of mutants and unaffected littermates. Only oocytes injected with cerebellar mRNA from mutants displayed statistically significant increases in responses to glutamate and kainate. In parallel morphological studies changes in the cerebellum of mutants were also observed. These consisted of a loss of Purkinje cells and an asymmetrical disarrangement of the granule cell layer of cerebellar cortex. Taken together, the physiological and morphological results suggest that alterations in glutamate/kainate receptors in the cerebellum are phenotypic manifestations of the Han-Wistar mutation. The results are consistent with the hypothesis that this mutant rat might serve as a model of glutamate/kainate excitotoxicity in the brain.

Postnatal development of voltage-dependent calcium channels in the mouse brain disclosed by the Xenopus oocyte assay.

We assessed the ontogeny of murine voltage-dependent calcium channels by extracting mRNA from brains of mice at different postnatal ages and injecting the mRNA into oocytes of the frog, Xenopus laevis. Voltage-dependent Ca2(+)-activated Cl- channels were measured to assess the presence of Ca2(+) channels. When compared with water-injected oocytes (controls), an increase in Ca2(+) channels was not detected until postnatal day 7. The number of Ca2+ channels peaked between 9 and 18 days and began to decline by 35 days. Bath application of barium, serotonin and the Ca2+ channel antagonist, verapamil, to mRNA-injected oocytes confirmed the presence of Ca2+ channels.

Caudate neurons respond to excitatory and inhibitory amino acids in early postnatal periods in the cat.

This study was designed to determine whether caudate neurons would respond to microphoretic application of glutamate and gamma-aminobutyric acid (GABA) in early postnatal periods in the cat. Extracellular recordings were performed in 175 neurons in developing kittens and 114 neurons in adult cats. At the earliest ages tested (1-10 days), caudate cells were excited by microphoretic application of glutamate and were inhibited by application of GABA. The results also indicated that caudate units have lower response thresholds to application of glutamate and GABA in early postnatal periods than in later periods. Since previous findings indicated that synaptically mediated inhibitory potentials develop during later postnatal periods in the cat, the present findings suggest that receptors for GABA may be capable of functioning before presynaptic endings make operational contacts.

Intracellular neurophysiological analysis reveals alterations in excitation in striatal neurons in aged rats.

Intracellular recordings were used to characterize the physiological changes underlying decreases in excitation observed in striatal neurons during the aging process. Rats were divided into 3 age groups: young (3-5 months), middle-aged (10-12 months) and aged (greater than 24 months). All experiments were performed in urethane-anesthetized rats. Recordings were obtained from 33 neurons in young, 17 in middle-aged and 20 in aged rats. When identified by intracellular injections of Lucifer yellow all recorded neurons were medium-sized spiny cells. Resting membrane potentials were at least -40 mV and action potentials greater than 35 mV. Postsynaptic responses were evoked by stimulation of frontal cortex. In all recorded neurons, regardless of age, excitatory postsynaptic potentials (EPSPs) could be evoked. However, the threshold currents for eliciting both EPSPs and synaptically driven action potentials were significantly higher in neurons obtained from aged rats than those recorded in the other two groups. Other changes in excitation in aged striatal neurons consisted of absence of spontaneously occurring EPSPs, higher current to induce firing by intracellular injections of depolarizing current and an inability of orthodromically induced action potentials to follow paired stimulation pulses to the cortex at short interpulse intervals. These data were interpreted to indicate that a combination of changes in synaptic connectivity and in membrane properties underlie the decreases in excitation. Together with our previous findings obtained from aged cats these results indicate that decreased neuronal excitability is a major effect of aging in the striatum.

Kynurenic acid antagonizes the excitatory postsynaptic potential elicited in neostriatal neurons in the in vitro slice of the rat.

Stimulation of corpus callosum in rat brain slices evoked an excitatory postsynaptic potential (EPSP) in neostriatal neurons. This EPSP is greatly reduced by exposing the slice to kynurenic acid (KY). The action of KY is reversed when the preparation is bathed in normal Ringer's solution. KY reduces the EPSP amplitude in a dose-dependent fashion, with 1 mM KY attenuating the potential by 86%. The effect is not due to a conductance change, since KY treatment does not alter the neuron's input resistance.

Dye-coupling in the neostriatum of the rat: I. Modulation by dopamine-depleting lesions.

Evidence from experiments performed in turtle and fish retina suggests that dopamine (DA) modulates the permeability of gap junctions. The present experiment was aimed at determining if DA has a similar role in the mammalian neostriatum. Adults rats received one of four treatments: unilateral electrolytic substantia nigra lesions, unilateral injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra, unilateral neocortical aspiration, or no treatment. After 3-5 weeks, neostriata from both sides of the brain were prepared for in vitro intracellular recordings. Recorded neurons (N approximately 150) were filled with Lucifer Yellow (LY), a low molecular weight dye that crosses gap junctions. In animals with electrolytic nigral lesions, dye-coupling in the ipsilateral neostriatum occurred after 38% of the intracellular injections. After 6-OHDA lesions, 19% of the injections produced dye-coupling in the ipsilateral neostriatum. This difference may have been accounted for by the fact that electrolytic lesions produced a greater degree of DA loss than 6-OHDA injections. Both of these percentages contrast with the very small percentage of dye-coupling found in intact animals or in animals with neocortical lesions. Dye-coupling occurred only between medium-sized spiny cells. No morphological differences between dye-coupled and non-dye-coupled cells were observed with light microscopy. Overall, passive and active electrophysiological properties of dye-coupled and single neurons were similar. The results suggest that DA may function in the neostriatum to control permeability of gap junctions.

Dye-coupling in the neostriatum of the rat: II. Decreased coupling between neurons during development.

Physiological and morphological evidence for coupling between neostriatal neurons was obtained from the developing rat. Intracellular injections of Lucifer Yellow-CH (LY) were made in rat neostriatal slices to study dye transfer (coupling) between neurons. The incidence of interneuronal coupling was 70% in early postnatal (P) periods and declined gradually to 10% in the adult. The number of neurons filled by a single intracellular injection also declined with age. LY injection into single neurons commonly marked aggregates of 4 to 6 cells in neonates. Single injections never produced more than one coupled pair in P20 or older rats. Neurons in which fast prepotentials (FPPs) could be evoked were consistently found to be dye-coupled. FPPs were resistant to collision with action potentials generated by intracellular current injection. When chemical synaptic transmission was blocked Mn2+, short-latency depolarizations (SLDs) could be evoked by extracellular stimulation. The SLDs were distinguished from chemical synaptic potentials by their "all or none" nature and by their insensitivity to changes in membrane potential. No SLDs were observed in adult neurons. FPPs and SLDs may be indicators of electronic transmission between coupled cells. The high incidence of coupling early in development might reflect intercellular communication that contributes to the differentiation and growth of neostriatal neurons.

GABAergic basal forebrain neurons project to the neocortex: the localization of glutamic acid decarboxylase and choline acetyltransferase in feline corticopetal neurons.

Our objective was to determine whether GABAergic and cholinergic basal forebrain neurons project to the neocortex. The retrograde connectivity marker wheat germ agglutinin lectin-bound horseradish peroxidase was injected into the neocortex of adult cats. Histo- and immunohistochemical methods were combined to label sequentially connectivity and transmitter markers (glutamic acid decarboxylase; choline acetyltransferase) in forebrain neurons. The labels of each marker were identified by correlative light and electron microscopy. Two principal types of doubly labeled neurons were demonstrated. The connectivity marker was colocalized with glutamic acid decarboxylase or choline acetyltransferase. The neurons were located in the basal forebrain. Their ultrastructural, cellular, and regional organization supported 2 conclusions. (1) GABAergic basal forebrain neurons project to the neocortex. This is important new morphological evidence for the origin of inhibitory neocortical afferents from a subcortical brain site. (2) The GABAergic and cholinergic basal forebrain neurons projecting to the neocortex exhibit remarkable structural similarities. The transmitter diversity of these intertwined neocortical afferents may be significant for the pathology and treatment of human neurological disorders such as Alzheimer's disease.

Ultrastructural alterations in caudate nucleus in aged cats.

These studies provide information on the changes in the ultrastructure in the caudate nucleus of aged cats. The major finding was that there was a decrease in the density of synapses in caudate neuropil. This decrease occurred in animals after 3 years of age and remained relatively constant in older animals. In conjunction with this change a population of unusually long synapses also occurred. These larger synaptic appositions were associated with enlarged spine heads. The caudate also showed a number of qualitative ultrastructural alterations. Many neurons contained accumulations of lipofuscin or lipopigment granules in aged animals. These inclusions occurred in both soma and dendrites of neurons and all types of glial cells. A unique configuration of collapsed agranular cisterns also was observed in aged animals. The present results indicate that decreases in synaptic density may be one morphological event underlying functional alterations observed in caudate neurons in aged cats.

Physiological and morphological characterization of striatal neurons transplanted into the striatum of adult rats.

Physiological and morphological properties of transplanted striatal neurons (TSNs) were examined in an in vitro slice preparation. Fetal striatal tissue (E13-14) was implanted as a dissociated cellular suspension into the striatum of adult rats. Intracellular records were obtained from TSNs 2-6 weeks after transplantation. TSNs exhibited biophysical, morphological, and synaptic properties characteristic of normal striatal neurons, despite the disruption involved in processing of the fetal tissue. Differences were observed, however, between the TSNs and host striatal neurons. TSNs consistently had higher input resistance values than host striatal neurons as determined by neuronal responses to intracellular current injection. Stimulation of adjacent host striatum elicited both excitatory and inhibitory postsynaptic potentials in TSNs. By contrast, the same stimuli elicited only excitatory responses in host striatal neurons. Morphologically, TSNs resembled host medium-size spiny neurons as demonstrated by intracellular injection of lucifer yellow. However, the complexity of dendritic branching and the density of spines on the dendrites were less than that observed for host striatal neurons. It was concluded that during the posttransplantation period studied, TSNs possess neuronal properties expected of developmentally immature striatal neurons.

The morphogenesis of glutamic acid decarboxylase in the neostriatum of the cat: neuronal and ultrastructural localization.

Correlative light and electron microscopic immunohistochemical methods were adapted for a descriptive analysis of the normal time course and pattern of expression and intraneuronal localization of the enzyme glutamic acid decarboxylase (GAD) in the neostriatum (Ns) of fetal, postnatal and adult cats. The differentiation of this synthesizing enzyme demonstrated the establishment of gamma-aminobutyric acid (GABA) transmitter identity in these neurons and their connections. The structural modifications of these GABAergic profiles revealed the morphogenesis of important inhibitory synaptic inputs in the Ns. The expression of GAD began during late fetal development and proceeded in a diagonal gradient from the first-formed ventrolateral putamen to the last-formed dorsomedial caudate nucleus. The frequency of GAD-positive elements increased with age particularly during the early postnatal period. After the initial expression of GAD, 3 interrelated processes contributed to its differentiation: (1) enzyme accumulation; (2) enzyme association with membranous organelles and (3) progressive elaboration of neuronal infrastructure. Synaptogenesis was both coincident and subsequent to GAD differentiation. Two principal types of GABAergic structures, cell bodies and axonal 'terminals', were evident from the initiation of GAD expression. The GABAergic cell bodies were polymorphic by and after the day of birth and consisted of ubiquitous medium sized cells (often having somatic and/or dendritic spines) and rare large sized cells (apparently aspiny and confined to a limited region of the Ns). The GABAergic axonal terminals changed from growth cone and prototerminal forms to mature bouton en passage and bouton terminaux forms establishing axosomatic and axodendritic contracts, having symmetric synaptic specializations and providing inputs to both medium- and large-sized GABAergic target neurons.

Sensory, motor and cognitive alterations in aged cats.

These experiments were designed to assess some of the sensory, motor and cognitive alterations that occur in aged cats. Three groups of cats (1-3, 5-9 and 11-16 years of age) were tested in four behavioral tasks to assess age-dependent changes in locomotor activity, fine motor coordination, reactivity to auditory stimuli and spatial reversal learning. In tests of locomotor activity, 11-16 year old cats displayed altered patterns of habituation compared to 1-3 and 5-9 year cats. There were no decrements in fine motor coordination in the 11-16 year cats as measured by their ability to traverse planks of varying width or by their scores on a neurological examination. The 11-16 and 5-9 year cats both displayed increased reactivity to auditory stimuli. On tests of spatial reversal learning, 11-16 year cats displayed superior performance compared to 5-9 or 1-3 year animals, making fewer errors and requiring fewer trials to reach criterion. These findings indicate that a series of age-related behavioral changes occurs in the cat. Some of these may be related to morphological and neurophysiological alterations in neurons in the caudate nucleus.

Aging reduces somatosensory responsiveness of caudate neurons in the awake cat.

Neuronal activity was recorded in 7 awake cats (3 animals 11-15 years and 4 animals 1-3 years of age) to determine if the ability of caudate neurons to process facial somatosensory information was impaired in aged animals. Proportionately fewer neurons responded to somatosensory stimuli and facial receptive fields were larger in the aged cats. In addition, proportionately fewer caudate neurons responded to cortical activation, there were fewer excitatory responses and spontaneous firing decreasing in aged cats. These results provide additional evidence that excitability of caudate neurons is reduced in aged cats.

Neurophysiological alterations in caudate neurons in aged cats.

These neurophysiological studies provide information on the alterations in functional capacity of neurons in the aging caudate nucleus (Cd) of the cat. The major finding is that there is a marked loss of excitation in the Cd during the aging process. This loss is most apparent in animals 11-14 years of age but is demonstrable in animals 6-7 years of age. Extracellular recording techniques were used to test the ability of Cd neurons to respond to activation of two of their major inputs, the precruciate cortex (CX) and the substantia nigra (SN). Types of responses that were evoked in both 1-3- and 11-14-year groups were similar and consisted of excitation, excitation followed by inhibition of action potentials or inhibition alone without preceding excitation. The frequency of occurrence of these responses was altered in the aged animals when either input was stimulated. In 1-3-year-old cats CX stimulation evoked initially excitatory responses in 75% of the cells tested while in 11-14-year-old cats excitatory responses occurred in 62% of the cells. When the SN was stimulated the decrease in initial excitation was greater (69% in 1-3- vs 35% in 11-14-year groups). In all aged animals but not in 1-3-year-old cats stimulation thresholds were higher (39-79%) for evoking excitatory responses than for evoking inhibitory responses. In order to assess synaptic security, the ability of Cd neurons to respond to iterative stimulation was determined. Distributions of the minimum interval necessary to evoke two excitatory responses were constructed. There was a marked increase in the proportion of longer intervals in the aged animals indicating that the synaptic response was less secure. There was a tendency for more of the responses in aged animals to have shorter latencies. This result was probably due to loss of less secure longer latency responses that are mediated via multisynaptic pathways. These findings indicate that there are functional changes in a population of Cd neurons in aged cats that impair their ability to process information.

The GABAergic striatonigral neurons of the cat: demonstration by double peroxidase labeling.

GABAergic striatonigral neurons were demonstrated in the adult cat by the specific double peroxidase labeling of a transmitter marker with an agranular appearance (GAD, the synthetic enzyme of GABA) and a connectivity marker with a granular appearance (WGA-HRP). Each marker was associated with different organelles confined to the perikaryal cytoplasm of neurons. GABAergic striatonigral neurons were of medium size, high frequency and wide location in the rostral caudate nucleus and putamen based on correlative light and electron microscopic identification. These cells had somatic and/or proximal dendritic spines and folded nuclear envelopes in some cases. They received GABAergic axosomatic and axodendritic inputs with symmetric synaptic specializations. They were also contacted by axosomatic, axodendritic and axospinous terminals with asymmetric synaptic specializations. These results indicate that the GABAergic striatonigral neurons are, for the most part, medium spiny cells that also emit intrastriatal axonal collaterals. Their intra- and extrastriatal axons mediate inhibitory postsynaptic influences on their targets. Their degeneration might contribute to the GABAergic deficits found in the basal ganglia in Huntington's disease.

Physiological and morphological analyses of ventral anterior and ventral lateral thalamic neurons in the cat.

Intracellular recordings were made from ventral anterior and ventral lateral (VA-VL) thalamic neurons in the cat. VA-VL neurons were tested for responsiveness to activation of cortical, pallidal and cerebellar afferents, and were identified morphologically by intracellular injection of HRP. Orthodromic activation of cortical and pallidal afferents produced primarily an initial inhibition (due in part to oligosynaptic circuitry) while activation of cerebellar afferents produced an initial excitation in the majority of neurons tested. Antidromic activation of thalamocortical relay neurons was observed in 32% of the neurons tested. Neurons showing short latency responses to activation of globus pallidus-entopeduncular nucleus and cerebellar peduncle were concentrated in the medical and ventral portions of the VA-VL complex, respectively. Neurons showing short latency responses to activation of the neocortex were located throughout the entire extent of the VA-VL complex. Only 3% of the neurons tested showed short-latency convergence of cortical, pallidal and cerebellar afferents. In contrast, 53% of neurons tested showed long latency triple convergence. Eight VA-VL neurons were stained intracellularly with HRP. Based on dendritic morphology, the labeled neurons were separated into two types: a stellate type with dendrites that spread radially from the soma, and a fusiform type with dendrites that were oriented mainly parallel to the long axis of the soma. Both types of neuron were aspiny although the dendrites of the stellate cells exhibited short appendages.

Basal forebrain neurons have axon collaterals that project to widely divergent cortical areas in the cat.

Basal forebrain neurons with axon collaterals that project to widely divergent cortical areas were identified using retrograde transport of two labels. A proportion of neurons in the basal forebrain have axon collaterals that project to both anterior (precruciate gyrus) and posterior (marginal and suprasylvian gyri) cortical areas or to medial (precruciate gyrus) and lateral (ectosylvian and anterior suprasylvian gyri) cortical areas. These branched fibers originate from cells located predominantly in the basal nucleus of Meynert. The existence of such neurons suggests that individual basal forebrain cells are capable of influencing widespread neocortical zones in the cat.