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Age-dependent modulation of short-term neuronal dynamics in the dorsal and ventral rat hippocampus

Brain aging is associated with alterations in the behavioral effectiveness to process information, due to mechanisms that are still largely unclear. Short-term neuronal activity dynamics are basic properties of local brain networks profoundly involved in neural information processing. In this study, we investigated the properties of short-term changes of excitatory synaptic transmission and neuronal excitation in the CA1 field of dorsal and ventral hippocampal slices from young adult and aged rats. We found that short-term synaptic plasticity (i.e. short-term dynamics of input to CA1 circuit) does not significantly differ between young and aged dorsal or ventral hippocampus. However, short-term dynamics of hippocampal output markedly differ between young and aged rats. Notably, age-dependent alterations in short-term neuronal dynamics were detected mainly in the dorsal hippocampus. Thus, the dorsal hippocampus of young rats can detect and facilitate transmission of 1-30 Hz input and depress transmission of higher-frequency input. In contrast, the aged dorsal hippocampus appears unable to transmit information in a frequency-dependent discriminatory manner. Furthermore, the amplification of steady-state output at frequencies < 40 Hz is considerably lower in the aged than young dorsal hippocampus. The aged ventral hippocampus did not show major alterations in short-term processing of neural information, though under conditions of intense afferent activation, neuronal output of the ventral hippocampus is depressed at steady-state more in aged than in young rats. These results suggest that aging is accompanied by alterations in neural information processing mainly in the dorsal hippocampus, which displays a narrower dynamic range of frequency-dependent transient changes in neuronal activity in aged compared with young adult rats. These alterations in short-term dynamics may relate to deficits in processing ongoing activity seen in aged individuals.


INTRODUCTION
Aging is a complex biological phenomenon which impacts brain function with various nonuniform ways (Samson and Barnes 2013). Previous evidence suggests that physiological aging of the brain is often associated with decline in some cognitive functions including everyday memory, sensory information processing and working memory (Salthouse et al. 1989;Craik and Jennings 1992;Kausler 1994;de Dieuleveult et al. 2017). Hippocampus is a brain structure particularly vulnerable during aging (Bettio et al. 2017), and normal aging people often show deficits in hippocampus-dependent memory (Rapp and Heindel 1994;Foster et al. 2012).
Also, hippocampus is prominently featured as a brain structure with particularly enhanced ability for plastic changes in synaptic transmission and local circuit dynamics (Bliss et al. 2007). Thus, hippocampus displays a very wide range of forms of activity-dependent neural plasticity, including phenomena of short-term synaptic plasticity. i.e. transient changes in synaptic transmission that last from tens of milliseconds to several minutes (Zucker and Regehr 2002; Regehr 2012), and long-term synaptic plasticity, i.e. changes in synaptic transmission that can last from about one hour to at least several weeks (Nicoll and Roche 2013). Classically, in studies of aging, a great attention has been given in forms of long-term changes in synaptic plasticity in the hippocampus, mainly because their conceptual similarities with timecorresponding hippocampus-dependent forms of memory (Martin and Morris 2002). Research during the past four decades has suggested that normal aging is accompanied by changes in synaptic plasticity in the hippocampus mainly expressed as a reduced ability for long-term potentiation of excitatory synaptic transmission; for recent reviews see (Foster 2002;Burke and Barnes 2006;Gray and Barnes 2015;Kumar 2020).
In addition to basic roles that phenomena of long-term synaptic plasticity appear to play in neural information processing and behavior (Dringenberg 2020), accumulating evidence shows that also short-term changes in synaptic effectiveness and neuronal activity, i.e. 4 frequency-dependent transient changes in synaptic transmission and neuronal excitation upon repeated activation, are deeply implicated in neural information processing, including temporal filtering, synaptic input diversification and dynamic gain control (Abbott and Regehr 2004;Jackman and Regehr 2017). Importantly, recent evidence shows that mechanisms that determine the properties of short-term neural dynamics (STND) play important roles in processing ongoing activity (Yang and Xu-Friedman 2015). Hence, considering the agingrelated deficits in sensory information processing (de Dieuleveult et al. 2017) one can hypothesize that short-term neuronal dynamics in the hippocampus may be altered during aging. Short-term changes in synaptic transmission and neuronal excitation have previously been examined in some aging studies, however, the results of these studies do not allow a consistent conclusion to be drawn, likely due to the limited range of experimental conditions under which STND were studied; see for instance (Landfield and Lynch 1977;Ouanounou et al. 1999;Ramsey et al. 2004). Therefore, the effects of aging on STND in the hippocampus have not yet sufficiently clarified.
Importantly, though hippocampus is susceptible to the aging process, it may not, however, be affected homogeneously along its longitudinal axis, as recent studies suggest (Yassa et al. 2011;Damoiseaux et al. 2016;Salami et al. 2016;Reichel et al. 2017;Dalton et al. 2019;Stark et al. 2020). Functional diversification along the longitudinal axis (called also septotemporal or dorsoventral axis) of the hippocampus is a concept supported by a substantial and rapidly growing body of evidence. According to this concept, different segments along the septotemporal axis of the hippocampus play discrete roles in several brain functions, most notably spatial cognition, memory and emotionality (Kheirbek et al. 2013;Bannerman et al. 2014;Strange et al. 2014;Tannenholz et al. 2014;Gulyaeva 2018). Interestingly, a recent study reports that intrahippocampal and extrahippocampal functional connectivity are reduced specifically in the anterior human hippocampus (that corresponds to the ventral hippocampus 5 in rodents) of older adults that appears to contribute significantly to age associated memory impairment (Stark et al. 2020). Hence, investigation of possible differences in the responsiveness to aging of distinct segments of the hippocampus along its long axis will help us to understand better the mechanisms underlying age-related declines in hippocampusdependent functions. However, the effects of aging on STND with respect to the longitudinal axis of the hippocampus has never been studied before. Notably, distinct segments along the septotemporal axis of the hippocampus display remarkably different properties of STND Kouvaros and Papatheodoropoulos 2016;Milior et al. 2016;Babiec et al. 2017;Papaleonidopoulos et al. 2017;Dubovyk and Manahan-Vaughan 2018; Koutsoumpa and Papatheodoropoulos 2019; Koutsoumpa and Papatheodoropoulos 2021), that may contribute to specific ways of information processing performed by discrete segments of the hippocampus. Possible differential age-associated alterations of STND along the hippocampus may contribute to specific cognitive impairments seen in elderly.
In the present study we examined STND applying short trains of stimulation pulses covering a wide range of frequencies (0.1 -100 Hz) in slices from the dorsal and the ventral hippocampus, prepared from young adult and aged rats. In addition, we examined STND under different levels of local circuit activation. We report that aging is accompanied by frequencydependent, segment-specific alterations in short-term neuronal excitation but not short-term synaptic plasticity. Possible implications of these findings are discussed.

RESULTS
Short-term changes in excitatory synaptic transmission (i.e., short-term synaptic plasticity) and short-term changes in neuronal excitation (i.e., short-term dynamics of neuronal output) in dorsal and ventral hippocampal slices were studied and compared between young and aged rats under similar experimental conditions. It has been previously shown that the magnitude of the first response in variable-frequency stimulation trains strongly influences the properties of 6 short-term changes in both synaptic transmission and neuronal excitation (Koutsoumpa and Papatheodoropoulos 2019). Specifically, regarding short-term synaptic plasticity, the magnitude of the first fEPSP in a ten-pulse train determines, to a great extent, whether the steady-state synaptic response to the stimulation train will be facilitation or depression.
Similarly, the intensity of stimulation current greatly affects the response of the local network to repeated activation. Notably, the dependence of short-term dynamics on the initial conditions of afferent stimulation is far more evident in the dorsal compared with the ventral hippocampus (Koutsoumpa and Papatheodoropoulos 2019). It is, therefore, imperative keeping the initial conditions similar when searching for the effects of a factor (the age, in the present study) on short-term synaptic/neuronal dynamics. We therefore proceeded to study short-term dynamics in the hippocampus of young and aged rats as follows: to study short-term changes in fEPSP, we applied frequency stimulation at three different stimulation current intensities, producing subthreshold, suprathreshold and submaximal responses, respectively; then, we averaged the responses collected under these three experimental conditions to obtain the final values for a particular slice. To study short-term changes in PS, we applied frequency stimulation at two different stimulation current intensities, producing PS with an amplitude less than 2 mV and PS with an amplitude equal or greater than 2 mV, respectively. Given that frequency stimulation trains starting with low and high amplitude PS mostly produce steady-state facilitation and depression, respectively, we decided to present results obtained under low and high intensity of stimulation separately. Only slices with similar initial fEPSPs and PSs from young and aged rats were included in the final analysis. Specifically, we studied short-term synaptic plasticity in young and aged rats that showed an initial fEPSP of 1.2 ± 0.09 mV/ms (41/27) and 1.2 ± 0.06 mV/ms (33/14) in dorsal and 1.17 ± 0.12 mV/ms (24/15) and 1.16 ± 0.08 mV/ms (24/15) in ventral hippocampal slices, respectively. Correspondingly, we studied short-term changes in neuronal excitation under conditions of afferent stimulation producing a PS less than 2 mV and 7 specifically 1.04 ± 0.05 mV (28/18) and 1.05 ± 0.05 mV (31/14) in the dorsal hippocampus and 1.05 ± 0.04 mV (27/18) and 1.04 ± 0.06 mV (24/15) in the ventral hippocampus of young and aged rats, respectively. Furthermore, we studied short-term changes in neuronal excitation under conditions of stimulation producing a PS equal to or greater than 2 mV; specifically 4.72 ± 0.36 mV (22/16) and 4.58 ± 0.34 mV (31/14) in the dorsal hippocampus and 4.34 ± 0.35 mV (17/12) and 3.89 ± 0.36 mV (21/15) in the ventral hippocampus of young and aged rats, respectively.

Short-term synaptic plasticity in dorsal and ventral hippocampus does not change during aging
Application of frequency stimulation in hippocampal slices from young rats showed that short- Wilk's Λ = 0.866) or the ventral hippocampus (MANOVA, F(100, 4258) = 1.08, p>0.1; Wilk's Λ = 0.836). Also, we did not find any significant age-related difference for the onset and steady-8 state response, in either the dorsal or the ventral hippocampus (independent t-test inside each segment of the hippocampus and at individual stimulation frequencies, p>0.05) (Fig. 4B). These results suggested that aging does not significantly affect short-term synaptic plasticity in either segment of the hippocampus.
Short-term dynamics of neuronal excitation changes during aging more in dorsal than in ventral hippocampus in a frequency-dependent manner As above described, we studied short-term modulation of neuronal excitation under two different conditions of intensity of afferent stimulation, relatively low and high, producing a PS < 2 mV and a PS ≥ 2 mV, respectively. Previous observations have shown that relatively low and high levels of initial excitation of hippocampal circuit produce mainly facilitation and depression of steady-state responses, respectively (Koutsoumpa and Papatheodoropoulos 2019). In keeping with these observations, we found that frequency stimulation of low intensity Comparing short-term dynamics between young and aged hippocampus, we found that the dorsal hippocampus of aged rats was unable to reach the score of facilitation displayed by young rats (Fig. 6A & Fig. 7A), therefore, aging significantly affected the frequency-dependent properties of STND in the dorsal hippocampus under these experimental conditions (MANOVA, F(90, 3883) = 2.91, p<0.0005; Wilk's Λ = 0.642). Specifically, facilitation of steady-state responses was significantly lower in the aged compared with young dorsal hippocampus (independent ttest at individual stimulation frequencies between 1 and 40 Hz, p<0.05) (Fig. 7B, Steady-state response). Furthermore, facilitation of the onset response was significantly lower in aged 9 compared with young dorsal hippocampus at stimulation frequencies 1-20 Hz (independent ttest at individual stimulation frequencies, p<0.05) (Fig. 7B, Onset response). Markedly, at higher stimulation frequencies (50-100 Hz) significant depression was observed in the young dorsal hippocampus (paired t-test, p<0.05), but not in ventral hippocampus, which continued to show facilitation (comparison between young and aged dorsal hippocampus, independent ttest, p<0.05). Steady-state depression produced in the dorsal hippocampus at high stimulation frequencies (50-100 Hz) was similar between young and aged rats (independent t-test, p>0.05).
Remarkably, aging did not significantly affect short-term dynamics of neuronal excitation in the

DISCUSSION
This study describes a detailed characterization of frequency-dependent short-term changes in synaptic transmission and neuronal excitation in the adult and aged dorsal and ventral CA1 hippocampal field. The main findings of the present study are the following: 1). Aging does not affect short-term synaptic plasticity either in the dorsal or the ventral hippocampus.
2). Aging is accompanied by frequency-dependent alterations in short-term dynamics of CA1 neuronal output, more in the dorsal than in ventral hippocampus.
3). The dorsal hippocampus of aged rats displays reduced short-term facilitation of steady-state neuronal output. 4). The dorsal hippocampus of aged rats displays reduced paired-pulse inhibition of excitation. 5). Under conditions of strong afferent activation, the ventral hippocampus of aged rats displays increased steady-state inhibition.
It is noted here that the effects of aging on short-term synaptic plasticity or short-term dynamics of neuronal excitation have never been studied before comparatively in the dorsal and ventral hippocampus. Past studies in the dorsal or middle part of the hippocampus have shown a tendency of young vs aged CA1 hippocampal circuit to show facilitation of excitatory synaptic transmission following relatively prolonged stimulation applied at a fixed frequency.
Specifically, long trains of Schaffer collateral stimulation (35-315 pulses) applied at the frequency of 7 Hz produced a greater facilitation of fEPSP in young than aged rats (Landfield et al. 1986). In another study fEPSP was facilitated in hippocampal slices from young but not aged rats, after 16 stimuli at 1 Hz (Ouanounou et al. 1999). Greater facilitation of intracellularly recorded EPSP was also observed in young vs aged hippocampus following the delivery of 11 long trains of suprathreshold but not subthreshold stimulation pulses (>40) at 7 Hz, (Thibault et al. 2001). In the present study, using a physiologically relevant stimulation consisting of only ten pulses and a wide range of stimulation frequencies, a pattern resembling normal firing activity of CA3 and CA1 pyramidal cells (Ranck 1973;Wong and Prince 1981;Johnston and Brown 1984;Fenton and Muller 1998), we did not observe any significant effect of age in shortterm synaptic dynamics, either in the dorsal or the ventral hippocampus. It seems therefore likely that age-dependent differences in short-term synaptic plasticity may occur under prolonged but not short periods of afferent activity in the hippocampus.
In contrast to the results on fEPSP, short-term dynamics of hippocampal output markedly differ between young and aged rats. Furthermore, age-dependent alteration in short-term neuronal dynamics were more pronounced in the dorsal than in ventral hippocampus. Previous studies have reported no age-related difference in paired-pulse facilitation of PS at inter-pulse intervals 4-200 ms (corresponding to 5-50Hz), and a greater facilitation of PS in young vs aged hippocampus only following long trains of stimulation pulses (≥150 pulses delivered at 12-15Hz), (Landfield and Lynch 1977;Landfield et al. 1978); however, in another study, prolonged stimulation at 7 Hz failed to show a significant effect of age on frequency potentiation or depression of PS (Landfield et al. 1986). In contrast to these results, we show that in young vs aged dorsal hippocampus, the paired-pulse facilitation of PS is greater at inter-pulse intervals corresponding to 1-20 Hz and paired-pulse inhibition of PS is greater at inter-pulse intervals corresponding to 50-100 Hz. Furthermore, the amplification of steady-state output (PS) at frequencies 1-40 Hz is significantly higher in the young than aged dorsal hippocampus. A higher facilitation of PS at 40 Hz has been previously observed in the CA3 field of young vs aged dorsal hippocampus (Villanueva-Castillo et al. 2017). In addition, we show that conditions of intense activation of the local network reveal a higher frequency-dependent inhibition of PS in aged vs young ventral hippocampus.
Several mechanisms that contribute to determining the properties of short-term dynamics can be involved to differentiate age-dependent short-term facilitation in neuronal excitability, including facilitation of excitatory synaptic transmission and mechanisms of disinhibition (Leung and Fu 1994). However, the age-associated differences in short-term neuronal excitability observed here could not be explained by short-term changes in excitatory synaptic transmission since aging does not affect short-term synaptic plasticity. An increased Conspicuously, however, frequency depression of PS is higher in the aged vs young ventral hippocampus, allowing us to hypothesize that the pattern of age-associated changes in GABAergic transmission in the ventral hippocampus may differ from that in the dorsal hippocampus. However, to the best of our knowledge, data on GABAergic transmission in the aged ventral hippocampus are still missing.

Possible implications
Regarding young adults, the results of the present study confirm previous observations that We conclude that aging is associated with reduced frequency-dependent dynamic range of the short-term changes in neuronal excitability more in the dorsal than in ventral hippocampus. This diversification may contribute to alterations in behaviorally observed information processing displayed by old individuals.

Animals and hippocampal slice preparation
Adult (3-4 months) and aged (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)  Specifically, animal was decapitated under deep anaesthesia with diethyl-ether, and the brain was removed after opening the cranium. The brain was placed in ice-cold ice-cold (2-4 o C) standard artificial cerebrospinal fluid (ACSF) containing, in mM: 124 NaCl, 4 KCl, 2 CaCl2 , 2 MgSO4 , 26 NaHCO3, 1.25 NaH2PO4 and 10 glucose. ACSF was equilibrated with 95% O2 and 5% CO 2 gas mixture at a pH=7.4. Then, the hippocampi were exposed and placed on the disc of a McIlwain tissue chopper. Transverse 500 µm-thick slices were prepared from the dorsal (septal) and the ventral (temporal) segment of the hippocampus extending between 1.0 mm and 3.0 mm from the DH and the VH end, as previously described. Immediately after their preparation, slices were transferred to an interface type recording chamber where they were maintained perfused constantly with standard medium of the same composition as above 15 described and at a rate of ~1.5 ml/min. Slices were continuously humidified with a mixed gas consisting of 95% O 2 and 5% CO 2 at a constant temperature of 300.5 o C. Tissue stimulation and recording started at least one and a half hours after their placement in the chamber.

Electrophysiological recordings, Data Processing and Analysis
Evoked field population potentials were recorded from the middle of the CA1 field following electrical stimulation of the Schaffer collaterals. Field excitatory postsynaptic potential (fEPSP) and population spike (PS) were recorded from the middle stratum radiatum and stratum pyramidale, respectively. Electrical stimulation consisted of current pulses of a fixed duration (100 μs) and variable amplitude (20 to 260 μA) applied through a bipolar platinum/iridium electrode with a wire diameter of 25 μm and an inter-wire distance of 100 μm (World Precision Instruments, USA). Recordings were performed using a 7 μm-thick carbon fiber (Kation Scientific, Minneapolis, USA) positioned 350 μm from the stimulation electrode. Most often, simultaneous recordings of fEPSP and PS were made from an individual slice. Baseline stimulation was delivered every thirty seconds using a current intensity that elicited a justsubthreshold fEPSP based on input-output curves between stimulation intensity and evoked responses. Short-term changes in fEPSP and PS were studied using a frequency stimulation protocol as previously described (Koutsoumpa and Papatheodoropoulos 2019). Specifically, the frequency stimulation protocol consisted of a sequence of ten consecutive pulses delivered at varying frequency between 0.1 and 100 Hz. The number of pulses falls into the range of naturally occurring spike trains in CA3 cells (Fenton and Muller 1998). Consecutive trains of pulses, delivered during the application of frequency stimulation paradigm, were separated by a two-minute interval to allow synapses to return to baseline level. Furthermore, stimulation trains of different frequency were applied at a random fashion during each experiment.
Frequency stimulation used to study short-term changes in fEPSP was applied at three intensities of stimulation current, while frequency stimulation used to study short-term changes 16 in PS was applied at two intensities of stimulation current. Specifically, we adjusted the stimulation current intensity to alternatively produce a subthreshold fEPSP of about 0.5 mV/ms, a suprathreshold fEPSP that evoked a PS of 0.5-1.0 mV, and a PS of 75% of its maximum amplitude. We call the three stimulation intensities that produced three different levels of local network activation subthreshold, suprathreshold and submaximal, respectively.
Signals were amplified 500 times, band-pass filtered at 0.5Hz-2kHz using Neurolog amplifiers (Digitimer Limited, UK), digitized at 10 kHz and stored on a computer disk for off-line analysis using the CED 1401-plus interface and the Signal6 software (Cambridge Electronic Design, Cambridge, UK). The fEPSP was quantified by the maximum slope of its initial rising phase; slope was measured in a time window of one millisecond immediately after the appearance of the presynaptic fiber volley. PS was quantified by its amplitude measured by the depression respectively) are marked by red and blue color respectively, as indicated in the color scale bar. Data in this figure are the same as in Fig. 3. B. Comparison of short-term synaptic plasticity between young and aged dorsal and ventral hippocampus. Percentages for fEPSPs produced by the 2nd pulse (onset response) and averages of fEPSP percentages produced by the 8th -10th pulses (steady-state responses), plotted as a function of stimulation frequency are shown. Frequency stimulation did not reveal any difference between young and aged rats, either for the onset or steady-state response in the dorsal or the ventral hippocampus. Data in this figure are the same as in Fig. 6. B. Comparison of short-term dynamics of neuronal excitation between young and aged dorsal and ventral hippocampus. Are shown percentages 30 for PSs produced by the 2 nd pulse (onset response) and averages of PS percentages produced by the 8 th -10 th pulses (steady-state responses), plotted as a function of stimulation frequency.
Asterisks indicate statistically significant differences between young and aged rats at p<0.05 (independent t-test at individual frequencies). Note that the facilitation induced by a wide range of stimulation frequencies in steady-state responses of the dorsal hippocampus is higher in young than in aged rats. Also, the second response in the dorsal hippocampus from young rats (onset response) facilitates at relatively low frequencies (< 40 Hz) and depressed at high stimulation frequency (> 40 Hz), while only facilitates in the dorsal hippocampus of aged individuals. Data in this figure are the same as in Fig. 8. B. Comparison of short-term dynamics of neuronal excitation between young and aged dorsal and ventral hippocampus. Are shown percentages for PSs produced by the 2 nd pulse (onset response) and averages of PS percentages produced 33 by the 8 th -10 th pulses (steady-state responses), plotted as a function of stimulation frequency.
Asterisks indicate statistically significant differences between young and aged rats at p<0.05 (independent t-test at individual frequencies). Note that strong stimulation intensity (i.e. evoking PS => 2 mV) applied at relatively high frequencies produces a greater depression of onset response in the dorsal hippocampus of young vs aged rats and a greater depression in the ventral hippocampus of aged vs young rats.
34 Figure 10. The range of short-term neuronal dynamics differs between young and aged hippocampus. 3D graphs illustrating the dynamic range of transient changes in synaptic input (fEPSP) and neuronal output (PS) in the dorsal and ventral hippocampus of young and aged rats. The graphs show the combined steady-state changes in fEPSP and PS as a function of stimulus frequency. Data regarding PS are obtained at a stimulation current intensity of low strength. The highest and the lowest value of each 3D plot (corresponding to intense facilitation and depression respectively) are marked by red and blue color respectively, as indicated in the color scale bar. Note that the dorsal hippocampus of young rats demonstrates higher scores of output facilitation compared with the dorsal hippocampus of aged rats, despite similar corresponding short-term changes in synaptic input in the two age groups.
In contrast, the dynamic range of steady-state responses in the ventral hippocampus did not significantly differ between young and aged individuals.