Action potential waveform variability limits multi-unit separation in freely behaving rats

Extracellular multi-unit recording is a widely used technique to study spontaneous and evoked neuronal activity in awake behaving animals. These recordings are done using either single-wire or multiwire electrodes such as tetrodes. In this study we have tested the ability of single-wire electrodes to discriminate activity from multiple neurons under conditions of varying noise and neuronal cell density. Using extracellular single-unit recording, coupled with iontophoresis to drive cell activity across a wide dynamic range, we studied spike waveform variability, and explored systematic differences in single-unit spike waveform within and between brain regions as well as the influence of signal-to-noise ratio (SNR) on the similarity of spike waveforms. We also modelled spike misclassification for a range of cell densities based on neuronal recordings obtained at different SNRs. Modelling predictions were confirmed by classifying spike waveforms from multiple cells with various SNRs using a leading commercial spike-sorting system. Our results show that for single-wire recordings, multiple units can only be reliably distinguished under conditions of high recording SNR (≥ 4) and low neuronal density (≈ 20,000/ mm(3)). Physiological and behavioural changes, as well as technical limitations typical of awake animal preparations, reduce the accuracy of single-channel spike classification, resulting in serious classification errors. For SNR <4, the probability of misclassifying spikes approaches 100% in many cases. Our results suggest that in studies where the SNR is low or neuronal density is high, separation of distinct units needs to be evaluated with great caution.     

Dynamics of neuronal activity in the posterior hypothalamus during alternating phases of the sleep-wake cycle

The dynamics of neuronal activity in the posterior hypothalamus in different phases of the sleep-wake cycle were investigated during experiments on free-ranging cats. The highest frequency discharges were found to occur in 89.3% of neurons belonging to this region during the stages of active wakefulness and emotionally influenced paradoxical sleep. These neurons become less active during restful wakefulness and the unemotional stage of paradoxical sleep; this reduced activity can be most clearly observed in the context of slow-wave sleep. It was found that 7.1% of test neurons discharged at the highest rate during the stage of active wakefulness. They did not achieve an activity level characteristic of active wakefulness during the period of paradoxical sleep, although activity level was higher than during other states. Only 3.6% of neurons followed the opposite pattern, with discharges succeeding more frequently in slow-wave sleep and activity reduced to an equal degree during wakefulness and paradoxical sleep. The neurophysiological mechanisms governing the sleep-wake cycle and how the posterior hypothalamus contributes to these mechanisms are discussed.I. S. Beritashvili Institute of Physiology, Academy of Sciences of Georgian SSR, Tbilisi. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 160–167, March–April, 1988.     

Microdialysis perfusion of orexin-A in the basal forebrain increases wakefulness in freely behaving rats

Recent work indicates that the orexin/hypocretin-containing neurons of the lateral hypothalamus are involved in control of REM sleep phenomena, but site-specific actions in control of wakefulness have been less studied. Orexin-containing neurons project to both brainstem and forebrain regions that are known to regulate sleep and wakefulness, including the field of cholinergic neurons in the basal forebrain (BF) that is implicated in regulation of wakefulness, and includes, in the rat, the horizontal limb of the diagonal band, the substantia innominata, and the magnocellular preoptic region. The present study used microdialysis perfusion of orexin-A directly in the cholinergic BF region of rat to test the hypothesis that orexin-A enhances W via a local action in the BF. A significant dose-dependent increase in W was produced by the perfusion of three doses of orexin-A in the BF (0.1, 1.0, and 10.0 microM), with 10.0 microM producing more than a 5-fold increase in wakefulness, which occupied 44% of the light (inactive) phase recording period. Orexin-A perfusion also produced a significant dose-dependent decrease in nonREM sleep, and a trend-level decrease in REM sleep. The results clearly demonstrate a potent capacity of orexin-A to induce wakefulness via a local action in the BF, and are consistent with previous work indicating that the BF cholinergic zone neurons have a critical role in the regulation of EEG activation and W. The data suggest further that orexin-A has a significant role in the regulation of arousal/wakefulness, in addition to the previously described role of orexin in the regulation and expression of REM sleep and REM sleep-related phenomena.     

Polyamines in the hypothalamus and hippocampus after the changes in corticosteroid levels in rats]

Adrenalectomy as well as single or multiple hydrocortisone and ACTH administration have been studied for their effect on putrescine, spermidine and spermine contents, ornithine decarboxylase and S-adenosyl-+methionine decarboxylase activities in the rat hypothalamic and hippocampal tissues and fraction of nervous endings. The data obtained prove complicated character of changes in polyamine metabolism in the studied structures when glucocorticoid level is disturbed. Hormone action is characterized by regional and intracellular specificity and depends upon the ratio of administration and the initial hormonal background of the organism. The significance of these changes for feedback regulation of ACTH secretion and metabolism of GABA is discussed.     

Detection of cold pain, cold allodynia and cold hyperalgesia in freely behaving rats

In mammals, somatosensory input activates feedback and feed-forward inhibitory circuits within the spinal cord dorsal horn to modulate sensory processing and thereby affecting sensory perception by the brain. Conventionally, feedback and feed-forward inhibitory activity evoked by somatosensory input to the dorsal horn is believed to be driven by glutamate, the principle excitatory neurotransmitter in primary afferent fibers. Substance P (SP), the prototypic neuropeptide released from primary afferent fibers to the dorsal horn, is regarded as a pain substance in the mammalian somatosensory system due to its action on nociceptive projection neurons. Here we report that endogenous SP drives a novel form of feed-forward inhibitory activity in the dorsal horn. The SP-driven feed-forward inhibitory activity is long-lasting and has a temporal phase distinct from glutamate-driven feed-forward inhibitory activity. Compromising SP-driven feed-forward inhibitory activity results in behavioral sensitization. Our findings reveal a fundamental role of SP in recruiting inhibitory activity for sensory processing, which may have important therapeutic implications in treating pathological pain conditions using SP receptors as targets.     

Effect of ionotropic glutamate receptors antagonists on the modifications in extracellular glutamate and aspartate levels during picrotoxin seizures: a microdialysis study in freely moving rats

Our previous studies have shown a local decrease in glutamate and aspartate levels during seizures, induced by picrotoxin microdialysis in the hippocampus of chronic freely moving rats. In this paper, we study the effect of continuous hippocampal microperfusion of the NMDA, AMPA and kainate glutamate receptor inhibitors 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine (MK-801); 6,7-dinitroquinoxaline-2,3-dione (DNQX), and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466). We also examine the action of L(-)-threo-3-hydroxyaspartic acid (THA), a glutamate and aspartate reuptake blocker, on the modification of extracellular glutamate and aspartate levels induced by picrotoxin, using the microdialysis method in freely moving rats. We found that changes in extracellular hippocampal concentrations in both amino acids are prevented by NMDA, AMPA and kainate receptor inhibitors. Seizures elicited under DNQX also induce a transient increase in aspartate extracellular levels coincident with seizure time. L(-)-threo-3-hydroxyaspartic acid increased the basal extracellular concentrations of both amino acids, but did not prevent the seizure-related decrease. Our results suggest that glutamate, the major neurotransmitter at the synaptic level, may also play an important role in non-synaptic transmission during seizures.     

Rapid changes in monoamine levels following administration of corticotropin-releasing factor or corticosterone are localized in the dorsomedial hypothalamus

Monoaminergic systems are important modulators of the neuroendocrine, autonomic, and behavioral responses to stress-related stimuli. The male roughskin newt (Taricha granulosa) was used as a model system to investigate the effects of corticotropin-releasing factor (CRF) or corticosterone administration on tissue concentrations of norepinephrine, epinephrine, dopamine, 3,4-dihydroxyphenylacetic acid, serotonin, and 5-hydroxyindoleacetic acid (5-HIAA) in microdissected brain areas. Intracerebroventricular infusion of 25 or 50 ng of CRF increased locomotor activity and site-specifically increased dopamine concentrations within the dorsomedial hypothalamus 30 min after treatment when compared to vehicle-treated controls. In further studies, male newts were treated as follows: (1) no injection, no handling, (2) saline injection, or (3) 10 microg corticosterone and then placed in a novel environment. Monoamine and monoamine metabolite concentrations were similar in the unhandled and saline-injected controls 20 min after treatment. In contrast, corticosterone-injected newts had elevated concentrations of dopamine, serotonin, and 5-HIAA in the dorsomedial hypothalamus (a region that contains dopamine- and serotonin-accumulating neuronal cell bodies in representatives of all vertebrate classes) but not in several other regions studied. These site-specific neurochemical effects parallel neurochemical changes observed in the dorsomedial hypothalamic nucleus of mammals following exposure to a variety of physical and psychological stress-related stimuli. Therefore, these changes may reflect highly conserved, site-specific neurochemical responses to stress and stress-related neurochemicals in vertebrates. Given the important role of the dorsomedial hypothalamus in neuroendocrine, autonomic, and behavioral responses to stress, and a proposed role for this region in fast-feedback effects of glucocorticoids on the hypothalamo-pituitary-adrenal axis, these stress-related monoaminergic changes are likely to have important physiological or behavioral consequences.     

Plasma growth hormone and thyrotropin responses to thyrotropin releasing hormone in freely behaving and urethane anesthetized rats.

Plasma GH and TSH responses to thyrotropin releasing hormone (TRH) were examined in freely behaving and urethane anesthetized rats. The i.v. administration of TRH (200ng/100g b.wt.) resulted in consistent elevations of plasma GH only in urethane anesthetized rats, while significant elevations of plasma TSH were similarly observed in both conditions. Results suggest that urethane influences plasma GH responses to TRH.     

Effects of neonatal administration of monosodium glutamate and castration on neurokinin A levels in the hypothalamus and anterior pituitary of rats.

The effects of neonatal administration of monosodium glutamate (MSG) and castration on hypothalamic and anterior pituitary levels of neurokinin A (NKA) were studied in male and female rats killed at 46 days of age. In male rats treated neonatally with MSG, body, anterior pituitary, testis, ventral prostate, and seminal vesicle weights and serum testosterone levels were significantly lower than in saline-injected controls. Hypothalamic NKA was significantly lower in MSG-treated male rats as compared with the controls, and no apparent changes were recorded in anterior pituitary NKA. Orchidectomy was followed by a significant decrease in hypothalamic NKA in saline controls, but not in MSG-treated rats. In female rats treated with MSG, there was a significant decrease in body, anterior pituitary, and ovarian weights, as compared with saline-injected controls, but no significant differences were observed in uterine weights and serum estradiol levels. Hypothalamic NKA was lower, although not significantly, in MSG-treated rats as compared with the respective controls, and no differences were recorded in anterior pituitary NKA levels. Ovariectomy was followed by a significant decrease in hypothalamic NKA in both MSG-treated and control rats, but NKA in the anterior pituitary was significantly increased after ovariectomy only in saline-treated controls, whereas MSG-treated females failed to show this response. It is concluded that neonatal MSG treatment resulted in a decrease of hypothalamic NKA, which was particularly pronounced in male rats without any significant change in anterior pituitary NKA levels. The response of hypothalamic NKA to castration and the response of anterior pituitary NKA to ovariectomy were also altered in MSG-treated rats; this may reflect a functional block of some neuroendocrine functions of the hypothalamus that resulted from the neuronal lesions induced by MSG.     

Role of Homer proteins in the maintenance of sleep-wake states

Sleep is an evolutionarily conserved process that is linked to diurnal cycles and normal daytime wakefulness. Healthy sleep and wakefulness are integral to a healthy lifestyle; this occurs when an organism is able to maintain long bouts of both sleep and wake. Homer proteins, which function as adaptors for group 1 metabotropic glutamate receptors, have been implicated in genetic studies of sleep in both Drosophila and mouse. Drosophila express a single Homer gene product that is upregulated during sleep. By contrast, vertebrates express Homer as both constitutive and immediate early gene (H1a) forms, and H1a is up-regulated during wakefulness. Genetic deletion of Homer in Drosophila results in fragmented sleep and in failure to sustain long bouts of sleep, even under increased sleep drive. However, deletion of Homer1a in mouse results in failure to sustain long bouts of wakefulness. Further evidence for the role of Homer1a in the maintenance of wake comes from the CREB alpha delta mutant mouse, which displays a reduced wake phenotype similar to the Homer1a knockout and fails to up-regulate Homer1a upon sleep loss. Homer1a is a gene whose expression is induced by CREB. Sustained behaviors of the sleep/wake cycle are created by molecular pathways that are distinct from those for arousal or short bouts, and implicate an evolutionarily-conserved role for Homer in sustaining these behaviors.     

Unit activity of the posterior hypothalamus during brain and skin temperature changes in unanesthetized rabbits

Changes in the firing rate of posterior hypothalmic neurons in response to local elevation of the brain temperature by 0.6–1.5°C and to a rise and fall (separately and simultaneously) of the skin temperature by 3–5°C were investigated in unanesthetized rabbits. Neurons responding selectively to changes in brain temperature and skin temperature and neurons responding to both temperature stimuli were found in the posterior hypothalamus. During combined stimulation the unit activity was changed much more than in response stimulus. It is concluded that the region of the posterior hypothalamus participates in the integration of impulses from central and peripheral temperature receptors.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 5, No. 5, pp. 490–496, September–October, 1973.     

On-line detection of sleep-wake states and application to produce intermittent hypoxia only in sleep in rats.

Sleep-disordered breathing is associated with adverse clinical consequences such as daytime sleepiness and hypertension. The mechanisms behind these associations have been studied in animal models, especially rats, but intermittent stimuli such as hypoxia have been applied without reference to sleep-wake states. To determine mechanisms underlying the adverse physiological consequences of stimuli associated with sleep-disordered breathing requires criteria for detection of sleep-wake states on-line to trigger stimuli only in sleep. This study aimed to develop such a system for freely behaving rats. Twelve rats with implanted electroencephalogram and neck electromyogram electrodes were studied in the light and dark phases. Electroencephalogram frequencies in the high (20-30 Hz) and low (2-4 Hz) frequency bands distinguished non-rapid eye movement (REM) sleep, whereas neck electromyogram distinguished REM. Using these parameters in a simple algorithm led to detection accuracies of 94.5 +/- 1.0 (SE) % for wakefulness, 96.2 +/- 0.8% for non-REM sleep, and 92.3 +/- 1.6% for REM compared with blinded human judgment. The algorithm was then used to trigger hypoxic stimuli only in sleep. Because frequency and amplitude analysis is readily performed using a variety of commercial systems, incorporation of these parameters into such an algorithm will facilitate studies investigating mechanisms underlying the physiological consequences of sleep-related respiratory stimuli in a fashion that more effectively models clinical disorders.     

Circadian integration of sleep-wake and feeding requires NPY receptor-expressing neurons in the mediobasal hypothalamus

Sleep and feeding rhythms are highly coordinated across the circadian cycle, but the brain sites responsible for this coordination are unknown. We examined the role of neuropeptide Y (NPY) receptor-expressing neurons in the mediobasal hypothalamus (MBH) in this process by injecting the targeted toxin, NPY-saporin (NPY-SAP), into the arcuate nucleus (Arc). NPY-SAP-lesioned rats were initially hyperphagic, became obese, exhibited sustained disruption of circadian feeding patterns, and had abnormal circadian distribution of sleep-wake patterns. Total amounts of rapid eye movement sleep (REMS) and non-REMS (NREMS) were not altered by NPY-SAP lesions, but a peak amount of REMS was permanently displaced to the dark period, and circadian variation in NREMS was eliminated. The phase reversal of REMS to the dark period by the lesion suggests that REMS timing is independently linked to the function of MBH NPY receptor-expressing neurons and is not dependent on NREMS pattern, which was altered but not phase reversed by the lesion. Sleep-wake patterns were altered in controls by restricting feeding to the light period, but were not altered in NPY-SAP rats by restricting feeding to either the light or dark period, indicating that disturbed sleep-wake patterns in lesioned rats were not secondary to changes in food intake. Sleep abnormalities persisted even after hyperphagia abated during the static phase of the lesion. Results suggest that the MBH is required for the essential task of integrating sleep-wake and feeding rhythms, a function that allows animals to accommodate changeable patterns of food availability. NPY receptor-expressing neurons are key components of this integrative function.     

Age-related changes in the glutamate metabolism of cerebral cortical slices from rats

The glutamate metabolism in cerebral cortical slices from young (12 weeks) and aged (100 weeks) rats was studied. A highly significant reduction of low affinity glutamate uptake and its metabolism in the aged rats by 14.5% of the amount of the young rats was observed (Pº0.001). When the ratio of the radioactivity of respective metabolite divided by the sum total radioactivity of overall glutamate metabolites was compared, the incorporation to aspartate was significantly small (P<0.01), while that of the CO₂ liberated and the GABA synthesized were increased (P<0.05). These results support the hypothesis that in cerebral cortical slices from aged rats the transamination of glutamate is suppressed and its decarboxylation is enhanced despite markedly reduced low affinity glutamate uptake into the cerebral cortex. The difference could be explained by the fact that cerebral cortical slices from aged rats are more vulnerable to anoxia than those from young rats when exposed during slice preparation.     

Rapid changes in plasticity across generations within an expanding cedar forest

We investigated the inter‐individual variation of phenotypic plasticity and its evolution across three generations within an expanding forest. Plasticity was assessed in situ from dendrochronological data as the response of radial growth to summer rainfall. A linear mixed model was used to account for spatial effects (environment and stand structure), temporal factors (stand dynamics) and the variation with age. Beyond these effects, our results reveal a significant inter‐individual variance of growth and plasticity within each generation. We also show that the mean values and variances of growth and plasticity changed significantly across generations, with different patterns for both traits. The possible environmental and genetic drivers of these changes are discussed. Contrasting with the trade‐off between stress tolerance and plasticity generally observed among populations, we detected a positive covariance at the individual level, which does not support the cost of plasticity hypothesis in this case.     

APM/CD13 and FOS in the hypothalamus of monosodium glutamate obese and food deprived rats

Protein (western blotting) and gene (PCR) expressions, catalytic activity of puromycin-insensitive membrane-bound neutral aminopeptidase (APM/CD13) and in situ regional distribution of CD13 and FOS immunoreactivity (ir) were evaluated in the hypothalamus of monosodium glutamate obese (MSG) and/or food deprived (FD) rats in order to investigate their possible interplay with metabolic functions. Variations in protein and gene expressions of CD13 relative to controls coincided in the hypothalamus of MSG and MSG-FD (decreased 2- to 17-fold). Compared with controls, the reduction of hypothalamic CD13 content reflected a negative balance in its regional distribution in the supraoptic, paraventricular, periventricular and arcuate nuclei. CD13-ir increased in the supraoptic nucleus in MSG (2.5-fold) and decreased in the paraventricular nucleus (2-fold) together with FOS-ir (1.5-fold) in FD. In MSG-FD, FOS-ir decreased (7-fold) in the paraventricular nucleus, while CD13-ir decreased in the periventricular (5.6-fold) and the arcuate (3.7-fold) nuclei. It was noteworthy that all these changes of CD13 were not related to catalytic activity of APM. Data suggested that hypothalamic CD13 plays a role in the regulation of energy metabolism not by means of APM enzyme activity.     

Immunohistochemistry of endogenous L-DOPA in the rat posterior hypothalamus

The aim of this work was to study L-DOPA-containing neuronal structures of the rat posterior and dorsal hypothalamus by means of immunohistochemistry using antiserum against glutaraldehyde conjugated L-DOPA. Aspects and distribution of L-DOPA immunoreaction among cells of the supramammillary nucleus and the A11, A13c and A13 cell groups are described and compared to dopamine immunoreactivity, mainly through a double colored labelling procedure employing a color modification of the DAB reaction by metallic ions. Differences between L-DOPA and dopamine stainings within cell groups as the presence of cells with predominant or exclusive L-DOPA coloration are tentatively explained under the light of previous findings using immunohistochemistry of catecholamines synthesizing enzymes and catecholamines histofluorescence.     

Immunohistochemistry of endogenous l-DOPA in the rat posterior hypothalamus

Summary The aim of this work was to study l-DOPA-containing neuronal structures of the rat posterior and dorsal hypothalamus by means of immunohistochemistry using antiserum against glutaraldehyde conjugated l-DOPA. Aspects and distribution of l-DOPA immunoreaction among cells of the supramammillary nucleus and the A11, A13c and A13 cell groups are described and compared to dopamine immunoreactivity, mainly through a double colored labelling procedure employing a color modification of the DAB reaction by metallic ions. Differences between l-DOPA and dopamine stainings within cell groups as the presence of cells with predominant or exclusive l-DOPA coloration are tentatively explained under the light of previous findings using immunohistochemistry of catecholamines synthesizing enzymes and catecholamines histofluorescence.